NZ728372A - Human-derived anti-huntingtin (htt) antibodies and uses thereof - Google Patents
Human-derived anti-huntingtin (htt) antibodies and uses thereof Download PDFInfo
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- NZ728372A NZ728372A NZ728372A NZ72837215A NZ728372A NZ 728372 A NZ728372 A NZ 728372A NZ 728372 A NZ728372 A NZ 728372A NZ 72837215 A NZ72837215 A NZ 72837215A NZ 728372 A NZ728372 A NZ 728372A
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Abstract
Provided are novel human-derived anti-huntingtin (HTT) antibodies and biotechnological derivatives thereof, preferably capable of binding mutated and/or aggregated HTT species and or fragments thereof, as well as methods related thereto. The human-derived anti-HTT antibodies and biotechnological derivatives can be used in pharmaceutical and diagnostic compositions for HTT targeted immunotherapy of Huntington Disease and diagnosis thereof.
Description
Human-derived anti-Huntingtin (HTT) antibodies and uses f FIELD OF THE INVENTION The present invention generally relates to antibody-based therapy ofHuntington's disease (HD) associated with Huntingtin (HTT). In particular, the present invention relates to novel molecules specifically binding to human HTT and/or antigens thereof, particularly human- derived antibodies as well as HTT—binding fragments, synthetic and biotechnological derivatives thereof, which are useful in the treatment of diseases and conditions induced by such pathogenic HTT isoforms.
In addition, the present invention relates to pharmaceutical and diagnostic itions comprising such HTT-binding molecules, antibodies and mimics thereof valuable both as a diagnostic tool to identify diseases and/or disorders associated with HTT aggregation and as a passive vaccination strategy for treating disorders related to diseases associated with HTT amyloidosis.
BACKGROUND OF THE INVENTION Huntington's disease (HD) is an autosomal dominant neurological amyloidogenic disease. 5 to 10 individuals per 100,000 individuals are ed with this autosomal disease. However, the prevalence in the US is much higher, studies have shown that under 200,000 US duals 50% have the risk of developing HD, in particular 30,000 ts are registered in the US while only 100,000 patients are registered worldwide.
HD, as shown in several studies, s from a trinucleotide CAG repeat expansion in the gtin (HTT) gene, in particular in exon 1 of the HTT gene located on some 4 (MacDonald et al., Cell 72, (1993), 971—983), which is translated into a polyglutamine (polyQ) stretch in the HTT n. HD occurs when the polyQ tract exceeds a threshold of 35-40 glutamine residues in length with a strong inverse correlation between repeat length and age- of—onset of disease. This polyQ stretch leads to a misfolding and aggregation ofHTT in several regions, 6. g. neurons and glial cells. With increasing age an lation of the HTT aggregates takes place leading to degeneration of the al GABA-ergic neurons and cortical dal neurons. Symptoms of the HTT misfolding and aggregation e involuntary movements, lack of motor coordination, depression, ive decline such as memory loss and/or dementia.
Since 1993 when the HD on was identified the understanding of the pathophysiology and molecular biology of the disease has cantly improved. Medicaments such as e. g.
Xenazine® (tetrabenazine, Lundbeck) a hexahydro-dimethoxy-benzoquinolizine derivative VMAT2 inhibitor had been designed for symptomatic treatment targeting involuntary muscle movements.
In addition, gene silencing approaches such as RNA interference (RNAi) have been suggested as potential therapies. In particular, the use ofsiRNA directed against HTT gene in a HD mouse model (R6/2) was shown to inhibit mutant HTT gene expression, see e.g. Warby et al., Am. J.
Hum Genet. 84 (2009), 351-366 and Olshina et al., Biological Chemistry 285 (2010), 21807- 21816. However, one limitation of this method lies in the difficulty to introduce sufficient amount of siRNA into the target cells or tissues as shown by e. g. Boudreau et al. (Brain Research 1338 (2010), 112-121). Furthermore this approach may face safety ities as a continued need for the expression of Huntingtin was suggested by gene deletions studies in animal models and cultured cells (Dragatsis et 05]., Nat. Genet. 26 , 300—306; er et al., Cell. 118 (2004), 127—138; Zuccato et al., Nat. Genet. 35 (2003), 76—83).
Therefore, there is a need for novel therapeutic strategies an ious and safe therapy of diseases associated with HTT aggregation which preferably directly interfere with amyloid formation by mutant HTT.
This technical problem is solved by the embodiments terized in the claims and described further below and illustrated in the Examples and s.
SUMMARY OF THE INVENTION The present invention provides anti-huntingtin (HTT) antibodies and equivalent HTT-binding molecules for use in the lactic or therapeutic treatment of diseases and conditions associated with HTT amyloidosis. More specifically, eutically useful human-derived antibodies as well as HTT-binding fragments, synthetic and biotechnological derivatives thereof that ize mutated and/or aggregated forms of HTT are provided.
In particular, experiments performed in accordance with the present invention were successful in the recombinant cloning and production of human-derived monoclonal HTT-specific antibodies which are specific for mutated and/or aggregated HTT species and/or nts thereof. The human ts being the source of the B cells from which the cDNA encoding the variable domain of human—derived monoclonal anti-HTT antibodies, tively, have been isolated, were healthy . However, in another embodiment of the present invention, the source of the B cells from which the human-derived monoclonal anti-HTT antibodies and the cDNA encoding their variable domain, respectively, might be isolated are HD patients carrying trinucleotide CAG repeat expansion in the HTT gene and being either symptom-free or displaying an unusually slow progressing or stable disease course or alternatively displaying l clinical features of Huntington's e. Furthermore, as demonstrated in the Examples, the antibodies of the present ion are capable of attenuating dendritic spine loss, improve behavioral performance during task-specific training and enhance sensorimotor ability in a mouse model of HD. Therefore, it is prudent to expect that the human monoclonal anti-HTT antibodies ofthe present invention and derivatives thereo fbesides being non—immunogenic also t a eutically beneficial effect in human.
As described in the background section, hitherto the pathogenesis of HD has been tried block by intracellular approaches such as RNA interference (RNAi); see also, e.g., Stanek et al., Human Gene Therapy 25 (2014), 461-474 for ing mutant Huntingtin by associated mediated RNA interference. With respect to an immunotherapeutic approach the intracellular expression of single-chain antibody fragments (scFv), z'.e. intrabodies which are devoid of the constant region of immunoglobulins such as of the IgG class has been explored in the last decade; see, e.g., supra and Butler et al., Prog Neurobiol. 97 (2012), for engineered intracellular scFV and single-domain (dAb; nanobody) antibody therapies to ract mutant gtin and related toxic intracellular proteins.
For example, Lecerf et al., Proc. Nat. Acad. Sci. 98 (2001), 769 describe a single-chain variable region fragment (scFv) antibody specific for the 17 N terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1 selected from a large human phage display. A corresponding scFv antibody, scFv-C4 comprising a lambda variable light (VL) chain (Kvam et al., PLoS One 4 (2009), e5727; GenBank accession number ACA53373) is described to have some protective effect in B6.CgHDR6/1 transgenic mice, a HD mouse model, which however ed both with severity of disease at time of injection. In order to improve the steady-level of the intrabody and to direct N-terminal htt exon 1 (httexl) protein fragments bound by scFv-C4 to the some for degradation in order to prevent them from aggregation the PEST signal sequence of Mouse Ornithine Decarboxylase (mODC) mODC has been fused to the scFv-C4 antibody; see Butler and Messer, PLoS One 6 (2011), e29l99. No in vivo experiments have been reported yet.
Also the group of Khoshnan et a]. was aiming at the development of intrabody-based therapeutics for HD and inter alia describe anti-huntingtin scFv antibodies d from mouse monoclonal dies binding the epitopes polyglutamine (polyQ), polyproline (polyP), and anti-C terminus and their effects upon intracellular expression on mutant huntingtin ation and toxicity; see, e.g, Ko et (11., Brain Research 56 (2001), 319-329, Khoshnan et al., Proc. Nat.
Acad. Sci 99 (2002), 1002—1007 and Legleiter et al., J. Biol. Chem. 284 (2009), 21647-21658 and their patent application US 2003/0232052 A1. In the US application, also a "human" scFv antibody denoted "hMW9" is described to have been ed from a human scFvs phage library using recombinant mutant huntingtin protein. However, in contrast to mouse monoclonal derived scFv MWl, MW2, MW7 and MW8 no ce data are provided for hMW9 which hitherto has also never been reported again.
Colby et (1]., Proc. Nat. Acad. Sci. 342 (2004), 901-912 describe the development of a human light chain variable domain (VL) intracellular antibody specific for the amino terminus of gtin via yeast surface display of a non-immune human antibody library. This single- domain intrabody consisting only of the lambda light chain domain of the original scFv was described to t huntingtin aggregation in a ree in vitro assay as well as in a mammalian cell culture model of HD; see also to corresponding ational application WC 2005/052002.
Again, no in vivo experiments have been reported yet.
Hence, apparently current intrabody based approaches either did not extend over cell-based assays or had not been proven to be successful in animal models ofHD yet, at least not in long term experiments. In particular, intrabodies reveal several limitations in—vz'vo such as their potential toxicity due to intracellular/intranuclear accumulation of intrabody-antigen complexes or the limited distribution of for example Viral delivery into large brain s in humans; see, e.g., Butler et al., Prog. iol. 97 (2012), 190-204 and Sothwell et al., J. Neurosci. 29 (2009), 13589-13602. Furthermore, a general drawback of intracellular approaches is the m of addressing the antibody and its encoding vector DNA, respectively, to the desired cells and, if exogenously applied the inconvenient administration regimen, for example intrastriatal injections; see, e.g, Snyder-Keller et al., athol. Exp. Neurol. 69 (2010), 1078—1085. In addition, general concerns with respect to gene therapy and the use of viral s remain.
In contrast, the experiments performed in accordance with the present invention demonstrate for the first time that fiJll-length IgG dies ed against different epitopes of huntingtin upon systemic administration can be successfully delivered to the brain (Example 24 and Figure 18) and that the antibodies of the present ion are capable of attenuating dendritic spine loss, improve behavioral performance during task—specific training and enhance sensorimotor ability in a mouse model ofHD (Example 34 and Figure 34).
Therefore, as illustrated in the Examples, the anti-HTT antibody or an HTT-binding fragment, synthetic or biotechnological derivative thereof is preferably of the IgG class, which as generally known and described herein comprises two identical variable heavy (VH) chain polypeptides and two cal le light (VL) chain polypeptides, and a constant region and domain, respectively, 2'. e. at least one or all of the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3). Put in other words, in one aspect of the present invention recombinantly expressed bivalent antibodies specific for gtin and aggregated forms, fragments, es and derivatives thereof are provided suitable for use in the treatment or in in viva diagnosis of gtin and disorders associated therewith, which are characterized by the presence of an immunoglobulin constant region. As bed herein r below the immunoglobulin may be of any class such as IgG, IgM, IgA IgG, or IgE and corresponding immunoglobulin subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl. Preferably however, the dy is of the human IgG subtype.
In addition, as also further explained herein, the human—derived antibodies of the present invention are characterized by comprising at least one or more CDR of human origin, 2'. e. being encoded by a cDNA derived from human memory B cells, and preferably wherein the VH and/or VL chain are of human memory B cell origin too. The constant region or any domain thereof if human may be of the same or different origin as the CDR(s) and the VH and/or VL chain, respectively.
In this t, unless stated otherwise or clear from context reference herein to the antibody of the present invention includes the human-derived antibodies illustrated in the Examples as well as HTT-binding nts, synthetic and biotechnological derivatives thereof.
As can be r noted form the prior art approaches of providing intrabodies derived from human scFvs phage library almost always schvs were obtained with a le light chain of Vlamda origin; see Kvam et al. and Colby et al., supra. In contrast, more than 90% of the derived antibodies ofthe present invention use a Vkappa light chain, which also applies to antibodies NI-302.31F11 and NI-302.35Cl illustrated in Example 24 to be capable of penetrating the brain upon ic administration and in Example 34 2.35C1) to have beneficial effects on behavioral performance and motor-related tasks of mice in a HD animal model. Therefore, it is tempting to ate that antibodies having a Vkappa light chain might have superior properties over antibodies having a light chain ofVlamda origin. Therefore, in a preferred embodiment of the antibody of the present invention the variable light chain is of Vkappa origin.
As illustrated in the Examples and Figures, the anti-HTT-antibody, HTT-binding fragment, synthetic and biotechnological variant thereof binds to different regions of the HTT exon 1 protein which shows the "toxic" alteration as described above, 126. the expanded, unstable trinucleotide repeat, as shown in the es. In particular, the antibody of the present invention recognizes a polyP-region, a polyP-region, the P-rich—region, the C terminal- region or the N—terminal region ofHTT exon 1 protein. The epitopes of the t dies illustrated in the Examples are summarized in Figure 20. As mentioned in the background section, HD occurs when the polyQ tract s a threshold of 35-40 glutamine residues in length due to an aggregation of HTT. Accordingly, as shown in Example 3, aggregated and soluble HTT exon 1 proteins with 21, 35 or 49 polyQ repeats were generated and the binding ofthe identified antibodies tested. In the following these constructs will be denoted HDX with X being the number of Qs, e. g. HTT exon 1 with 21 polyQ repeats will be denoted HD2l.
Therefore, unless specifically indicated otherwise the term HTT means HTT exonl and the soluble HTT refers to the corresponding GST-fusion proteins.
In a preferred embodiment of the present invention, the anti-HTT antibody or HTT-binding fragment, tic or biotechnological derivative thereof is capable of preferentially binding aggregated or misfolded forms of HTT. As described in e. g. Legleiter et al., JBC 285 (19) (2010), 14777—14790 and demonstrated in the es the aggregation of HDX proteins in terms of speed and seize increases with the number of Qs.
In a particularly preferred embodiment ofthe present invention, the anti-HTT antibody or HTT- binding fragment, synthetic or biotechnological derivative thereof demonstrates the immunological binding characteristics of an antibody characterized by any one of the variable regions VH and/or VL as set forth in Fig. 1. Preferably, the variable region of the antibody comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable regions, 1'. e. pair of VH and VL chain as set forth in Fig. 1A to lAU, wherein one or more amino acid substitutions are ted as long as binding specificity of the resultant antibody compared to the subject antibody comprising the corresponding pair of VH and VL chain as set forth in Fig. 1A to lAU as illustrated in the Examples, 6.g. as ized in Figure remains unaffected in kind, 1'. e. epitope specificity and ECso values in the same order of magnitude for the indicated antigen, preferably in the range of at least 50%, more preferably 25% and most preferably at least 10% identical value. Preferably, one, two or all three CDRs of the VH and VL chain contain at least one amino acid at a corresponding position which is conserved (zle. being the same or a conservative substitute amino acid) in at least about 20%, preferably about 40%, more preferably about 50% and most preferably about 75% in the VH and VL chain amino acid sequences, respectively, of the subject antibodies which recognize the same type of HTT epitope, z'.e. , P-rich, C-terminus or N—terminus. For example, sequence alignment of the subject antibodies reveals the predominant presence of one or two nes (Y) in CDRHl; see Figure 36. Similar conserved amino acid can be identified in the other CDRs as well.
In a further ment of the present invention, the anti-HTT antibody or HTT-binding fragment, synthetic or hnological tive thereof is a bispecific antibody. Thus, the antibody ofthe present invention may be capable of recognizing at least two distinct epitopes either on the same or on different antigens. For example, while a first n-binding site, Le. le domain may be specific for HTT and preferably comprises a variable region of any one of the t antibodies illustrated in the appended Examples and Figures, the second antigen—binding site may be specific for a different, preferably also neurotoxic n and comprise a variable region of corresponding antibody. Hence, protein misfolding and aggregation is a major rk of neurodegenerative disorders such as Alzheimer's disease (AD), son's disease (PD) and HD. Tough until recently, the consensus was that each aggregation—prone protein was characteristic of each disorder [a—synuclein (d-syn)/PD, mutant huntingtin (HTT)/HD, Tau and amyloid beta peptide/AD], growing evidence tes that aggregation- prone proteins can actually co-aggregate and modify each other's or and toxicity, suggesting that this process may also bute to the p in clinical symptoms across different diseases; see, e.g., for co-aggregation of a-syn and mutant HTT Pocas et £21., Hum. Mol. Genet. 24 (2015), 1898-1907.
Thus, in one embodiment of the present invention the anti-HTT antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof is a bispecific antibody which is capable ofbinding HTT and a protein associated with a neurodegenerative disorder, in particular in the brain, ably selected from the group consisting of d—synuclein, Tau, amyloid beta peptide, SODl, C9orf72, and TDP-43; see, e.g., Blokhuis et al., Acta Neuropathol. 125 (2013), 777—794. Human—derived monoclonal antibodies against the mentioned proteins are known in the art; see, e.g., international application W02008/081008 for beta dy, WO2010/069603 for anti-(x-synuclein antibody; WO2012/049570 for anti-tau antibody; W02012/080518 for anti-SOD] antibody; W02012/113775 for anti-ankyrin antibody; /061163 for anti-TDP-43 antibody and European patent application EP 14 187 180.6 and its subsequent international application for C9orf72. Bi- and multispecific antibodies can be generated by methods well known in the art, for example by chemical recombination of monoclonal immunoglobulin G1 nts as described, e. g., by Brennan et al., Science. 229 (1985), 81-83, or recombinant simultaneously co-expression of the appropriate heavy and light chain and corresponding pairing; see, e.g., Lewis et al., Nature Biotechnology 32 (2014), 191—198; for review see, e. g., Kontermann, mAbs 4 (2012), 182-197 and Kontermann and Brinkmann, Drug ery Today 20 (2015), 838—847.
Alternatively, or in addition the bi- or multi-specific dy comprises at least a first and second antigen-binding site, :16. variable domain specific for two distinct epitopes of HTT, preferably wherein one or both variable regions are derived from any one of the subject antibodies illustrated in the appended Examples and Figures, and as further bed herein. Thus, in a preferred embodiment the bispeciflc antibody ofthe present invention comprises two binding sites/domains of an antibody which recognizes a polyP-region, a polyQ/polyP-region, the P—rich-region, the C terminal-region, the N- terminal region or a conformational epitope of HTT exon 1 protein. The epitopes of the t antibodies illustrated in the Examples are ized in Figure 20. Accordingly, in one embodiment the ific antibody of the present invention recognizes at least two different es depicted in Figure 20 and has the combined binding specificities of the e subject dy, respectively.
The antigen-binding fragment of any one the subject antibodies disclosed herein can be a single chain Fv nt, an F(ab') fragment, an F(ab) fragment, and an F(ab')2 fragment, or any other antigen-binding fragment. However, as mentioned in a ularly preferred embodiment, the antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof is a human IgG isotype dy and comprises at least part of the constant region. Alternatively, the antibody is a chimeric human-rodent or rodentized antibody such as murine or murinized, rat or ratinized antibody, the rodent versions being particularly useful for diagnostic methods and studies in animals.
Furthermore, the present invention relates to compositions comprising the antibody of the present invention or antigen-binding fragment, synthetic or biotechnological derivative thereof and to immunotherapeutic and diagnostic methods using such compositions in the prevention, diagnosis or treatment of diseases and/or disorders associated with HTT amyloidosis, wherein an effective amount of the composition is administered to a patient in need thereof.
The present invention also relates to polynucleotides encoding at least a variable region of an immunoglobulin chain of the antibody of the invention. Preferably, said variable region ses at least one mentarity determining region (CDR) of the VH and/or VL of the le region as set forth in Fig. l. ably, the polynucleotide is a cDNA.
Accordingly, the present invention also encompasses vectors comprising said polynucleotides and host cells transformed ith as well as their use for the production of an antibody and equivalent binding molecules which are specific for HTT and preferably are capable of binding mutated and/or ated HTT species or fragments thereof. Means and methods for the inant production of antibodies and mimics thereof as well as methods of screening for competing binding molecules, which may or may not be antibodies, are known in the art.
However, as described herein, in particular with respect to therapeutic applications in human the dy of the present invention is a human antibody in the sense that ation of said antibody is substantially free of an immune response directed against such antibody otherwise ed for chimeric and even humanized antibodies. Hence, the present invention also relates to the use of the cDNA, vector and host cell bed herein and illustrated in the Examples for the production of an anti-HTT antibody, in particular human-derived anti-HTT antibody or a biotechnological derivative thereof.
Furthermore, disclosed herein are compositions and methods that can be used to identify HTT, in ular mutated and/or aggregated HTT species or fragments in vitro, 6.5;. in samples and/or in vivo. The disclosed anti-HTT antibodies and binding nts thereof can be used to screen human blood, plasma, serum, saliva, peritoneal fluid, cerebrospinal fluid ("CSF"), and urine for the presence of HTT and/or mutated and/or ated HTT species or fragments thereof in samples, for example, by using based or surface adapted assay. In one embodiment the present invention relates to a method of diagnosing or monitoring the progression of a disease and/or disorder related to mutated and/or aggregated HTT species or fragments thereof in a subject, the method comprising determining the presence of mutated, and/or ated HTT species or fragments in a sample from the subject to be diagnosed with at least one antibody of the present invention or an HTT-binding molecule and/or binding molecules for mutated and/or aggregated HTT species or fragments having substantially the same binding specificities of any one thereof, wherein the presence of mutated and/or aggregated HTT species or fragments is indicative of the disorder.
Accordingly, the present invention also relates to a method of preparing a ceutical composition for use in the treatment of a disorder associated with or caused by HTT aggregates, the method comprising: (a) expressing the cDNA of the present invention and/or culturing the host cell of the present ion under appropriate culture conditions suitable for the tion of the anti-HTT antibody, in particular human-derived anti-HTT antibody or a biotechnological derivative thereof; (b) purifying the antibody, biotechnological derivative or globulin chain(s) thereof from a reaction mixture and the culture, respectively, to pharmaceutical grade; (c) admixing the antibody or biotechnological derivative thereof with a pharmaceutically acceptable carrier.
Furthermore, in one ment of the present invention the anti-HTT antibodies and HTT- binding molecules comprising at least one CDR of an antibody of the present invention are provided for the ation of a composition for in viva detection (also called in viva imaging) of or targeting a therapeutic and/or diagnostic agent to HTT, in particular mutated and/or aggregated HTT species or fragments in the human or animal body. The methods and compositions sed herein can aid in diseases and/or disorders associated with HTT aggregation or amyloidosis and terized, e.g., by the occurrence of aggregated forms of HTT and can be used to monitor disease ssion and therapeutic y of the therapy ed to the subject, for example in in vivo imaging related diagnostic methods. In one embodiment the in vivo ion (imaging) comprises scintigraphy, positron emission tomography (PET), single photon emission aphy (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).
Hence, it is a particular object of the t invention to provide methods for treating, diagnosing or preventing a disease and/or disorder associated with HTT amyloidosis. The methods comprise administering an effective concentration of a preferably human antibody or dy derivative to the subject where the antibody s HTT or fragments thereof, preferably mutated and/or aggregated or misfolded HTT s or fragments thereof.
In a further aspect the t invention provides a peptide having an e ofHTT, preferably of mutated and/or aggregated HTT species or fragments thereof specifically recognized by an antibody ofthe present invention. Said peptide comprises or consists of an amino acid sequence as indicated below in the detailed description and in the Examples or a modified sequence thereof in which one or more amino acids are substituted, deleted and/or added. Additionally, the present invention provides a method for diagnosing diseases and/or disorders associated with HTT amyloidosis in a subject, comprising a step of determining the presence of an antibody that binds to said peptide in a ical sample of said subject.
Further embodiments of the present invention will be apparent from the description and es that follow.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1: Amino acid sequences of the variable s of human antibodies NI-302.33Cl 1, N1- 302.63F3, .35Cl, NI-302.31Fl l, NI-302.2A2, NI-302.6N9, NI-302.74Cl l, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI- 302.37C12, NI-302.55D8, NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9, .15E8, NI-302.15D3, NI-302.64E5, NI-302.7D8, NI-302.72F10, NI-302.12H2, NI-302.8M1 and .4A6. Framework (FR) and complementarity determining regions (CDRs) are indicated with the CDRs being underlined. The Kabat numbering scheme was used (cf. https://www.bioinf.org.uk/abs/).
Fig. 2: Characterization ofHuntingtin (HTT) exon 1 proteins and aggregates. (A) Cloning of GST-HttEx1Q21 (GST-HD21), GST-HttEx1Q35 (GST-HD35) and GST-HttEx1Q49 (GST-HD49) expression constructs; (B) Coomassie dye staining upon GE of purified GST only (lane 1), GST-HttExole2l (GST-HD21, lane 2), GST- HttExole35 (GST-HD35, lane 3) and GST-HttExonl Q49 (GST-HD49, lane 4) proteins showing good purity but also some additional bands; (C) Characterization of in vitro HD21, HD35 and HD49 time-resolved in vitro aggregation ons by dot- blot (left) and filter retardation is (right) with polyclonal HD-l antibody as ion antibody. Aggregation reactions ofHD35 at 24 hours or HD49 reactions after 3 hours show aggregates larger than the pore size of 0.2 um able by HD-l in the filter ation assay analysis; (D) Characterization of in vitro HD35 and HD49 preparations by electron microscopy. Aggregation reactions of HD35 after 24 hours [A, E] or HD49 reactions after 1 hour [B, F], 3 hours [C, G] or 24 hours [B, H]. ew pictures [A—D] with l’OOOX magnification and detailed structures [E-H] at 66'000X magnification.
Fig. 3: Characterization of the binding affinity of anti-polyP domain-binding antibody NI-302.33Cll. (A) NI-302.33Cll binding affinity for different HTT species determined by direct ELISA; (B) NI-302.33Cll ECso determinations for ated HD49 (o), aggregated HD21 (I), soluble GST-HD49 (A) and GST-HDZI (V) Htt Exon 1 proteins using direct ELISA. NI-302.33C11 antibody binds with similar ECso values to all four species; and (C) .33C11 g analysis to HTT aggregates on in vitro HD21, HD35 and HD49 time—resolved in vitro aggregation reactions by dot-blot (left) and filter retardation assay (right) with preferential binding to later (aggregated) ons of HD35 and HD49 in the dot-blot assays and aggregates of HD35 and HD49 in the filter retardation assay.
Fig. 4: Determination of NI-302.33Cll antibody binding epitope by scan of overlapping peptides. At the top: pepscan image after NI-302.33Cll antibody hybridization.
Below: graphical overviews ofpeptides sequences bound by NI-302.33Cll antibody.
Overlapping amino acids between peptides (putative binding epitope) being recognized by the NI- 302.33Cll antibody are highlighted in bold in the consensus sequences. The HRP-conjugated donkey anti-human IgG Fcy detection antibody alone does not bind any linear huntingtin e.
Fig. 5: NI-302.33Cll binds to the polyP-domain of HTT. ECso determinations for GSTHD49 (o), BSA-coupled P-rich domain peptide (O), BSA—coupled C-terminal e (I) or BSA-coupled polyP peptide (A) using direct ELISA.
Fig. 6: Characterization of the purity and integrity as well as the binding city of NI-302.33Cll antibody. SDS-PAGE analysis followed by Coomassie staining of 2 and 10 ug recombinant human NI-302.33C11 anti-polyP domain antibody.
Fig. 7: terization of binding affinity of roline—rich domain antibody N1302.63F3.
(A) NI-302.63F3 binding affinity for different HTT species determined by direct ELISA; (B) NI-302.63F3 ECso determinations for aggregated HD49 (O), aggregated HD21 (I), soluble GST-HD49 (A) and GST-HD21 (V) Htt Exon 1 proteins using direct ELISA. NI-302.63F3 antibody has a similar ECso values to all four species; (C) Characterization of antibody NI—302.63F3 on in vitro HD21, HD35 and HD49 time- resolved in vitro aggregation reactions by dot-blot (left) and filter retardation assay (right) with preferential g to huntingtin with expanded polyQ tracts HD35) in the dot-blot assays and aggregates of HD35 and HD49 in the filter retardation assay.
Fig. 8: Determination of NI-302.63F3 antibody g epitope by scan of overlapping es. At the top: pepscan image after NI—302.63F3 antibody hybridization. Below: graphical overviews of peptides sequences bound by NI-302.63F3 antibody.
Overlapping amino acids between peptides (putative binding epitope) being recognized by the NI-302.63F3 antibody are highlighted in bold in the consensus sequences. The HRP-conjugated donkey anti-human IgG Fey ion antibody alone does not bind any linear huntingtin peptide.
Fig. 9: NI-302.63F3 binds to the P—rich domain of HTT. ECso determinations for GST-HD49 (o), BSA-coupled P-rich domain peptide (0), BSA-coupled C-terminal peptide (I) or BSA-coupled polyP e (A) using direct ELISA.
Fig. 10: Characterization of the purity and integrity as well as the binding specificity of .63F3 antibody. SDS-PAGE analysis followed by Coomassie staining of 2 and 10 ug inant human NI- 302.63F3 anti-proline-rich domain antibody.
Fig. 11: Characterization of the binding affinity of -terminal domain-binding antibody NI 302.35Cl. (A) NI-302.35Cl binding affinity for different HTT species determined by direct ELISA; (B) NI-302.35Cl ECso determinations for aggregated HD49 (O), aggregated HD21 (I), soluble 49 (A) and GST-HD21 (V) Htt Exon 1 proteins using direct ELISA; (C) Characterization ofantibody NI-302.35C1 on in vitro HD2l, HD35 and HD49 time-resolved in vitro aggregation reactions by dot-blot (left) and filter retardation assay (right) with preferential binding to later (aggregated) reactions of HD35 and HD49 in the dot-blot assays and aggregates ofHD35 and HD49 in the filter ation assay.
Fig. 12: NI—302.35Cl binds to the BSA-coupled C-terminal domain peptide of HTT. ECSO determinations for GST-HD49 (o), BSA—coupled P-rich domain peptide (0), BSA- coupled C-terminal peptide (I) or BSA-coupled polyP peptide (A) using direct ELISA.
Fig. 13: Characterization of the purity and integrity as well as the binding specificity of NI-302.35Cl antibody. SDS-PAGE analysis followed by sie staining of 2 and ug recombinant human NI-302 anti-C-terminal domain antibody.
Fig. 14: Characterization ofbinding affinity of anti-N—terminal domain antibody N1302.15E8.
(A) NI-302. 15E8 binding affinity for different HTT species determined by direct ELISA; (B) NI-302.15E8 ECso determinations for aggregated HD49 (O), ated HD21 (I), e GST-HD49 (A) and GST-HD21 (V) Htt Exon 1 proteins using direct ELISA. NI-302. 15E8 antibody has a higher affinity binding ECso values to non- aggregated s.
Fig. 15: NI-302.15E8 binds to the BSA-coupled N—terminal domain peptide of HTT. ECso determinations for GST-HD49 (o), BSA-coupled inal peptide (V) BSA- coupled P-rich domain peptide (6), BSA—coupled C-terminal peptide (I) or BSA- coupled polyP peptide (A) using direct ELISA.
Fig. 16: Target specificity analysis by direct ELISA. NI-302 antibodies (A) .33Cl l, (B) .63F3, and (C) NI-302.35Cl and (D) NI-302.15E8 do not bind unrelated aggregating protein targets as shown in the binding specificity analysis by direct ELISA.
Fig. 17: Spine density is significantly d in hippocampal slice cultures of Tg(HDexonl)62pr/1J enic mice compared to non—transgenic littermates. (A- D) Overview of GFP positive hippocampal s of non-transgenic littermates (A, C) vs. Tg(HDexonl)62pr/1J mice (B, D), showing a single dendrite with the dual spines at higher magnification (C, D). (E) Significant reduction of tic spine density in transgenic vs. wildtype animals (n=3-7 slices per group from 2 wt or 3 transgenic animals). (F) ation of dendritic spine density loss by antibodies NI- 302.11Fll and 302.63F3 in slices oftransgenic mice. (n=8—l3 slices per group from a total of 12 transgenic s). Data represent the mean i SEM. *p<0.05 (MWU), # p=0.05.
Fig. 18: Penetration of NI-302 antibodies in the brain of R6/l animal model. (A) Mean NI- 302.31Fll (o) and NI-302.35Cl (I) plasma and brain drug levels in R6/l transgenic animals after a single intraperitoneal injection of 50mg/kg. Data represent the mean :: SEM. n=3 for each group; (B) Plasma and brain drug levels of individual mice after a single dose of 50 mg/kg.
Fig. 19: EC50 determinations of human-derived HTT dies for aggregated HD49 (O), aggregated HDZl (I), soluble GST-HD49 (A) and GST-HD21 (V) Htt Exon 1 ns using direct ELISA. Some antibodies (e.g. NI-302.37C12 (I), .55D8 (J), NI-302.11A4 (F) or NI-302.22H9 (G)) seem to have preferred binding to uncut GST-HTT protein suggesting that these antibodies preferentially recognize uncut soluble GST-HD constructs whereas some antibodies (e.g. NI-302.74Cll (C) or NI- 302.71F6 (M)) showed high affinity binding with similar EC—values to all HTT preparation suggesting that they bind to an e that is similar d in aggregated and uncut HTT exon 1 constructs in the ELISA assay.
Fig. 20: Characterization of binding affinity by direct ELISA. Binding affinity to the different HTT ns of human-derived HTT-specific antibodies Fig. 21: Characterization of antibody NI—302.44D7, NI-302.37C12, NI—302.15F9 and NI- 302.71F6 on in vitro HD2l, HD35 and HD49 time-resolved in vitro aggregation reactions by dot-blot (left) and filter retardation assay (right) with preferential binding in particular NI-302.15F9 and NI-302.71F6 to later (aggregated) reactions of HD35 and HD49 in the dot-blot assays and SDS stable aggregates of HD35 and HD49 in the filter retardation assay.
Fig. 22: Target specificity analysis by direct ELISA. NI-302 antibodies (A) NI-302.31Fl l, (B) N1-302.6N9, (C) NI-302.46C9, (D) NI-302.8Fl, (E) .2A2, (F) N1-302.74C11, (G) NI-302.15F9, (H) NI—302.39Gl2, (I) NI-302.11A4, (J) NI—302.22H9, (K) NI- D7, (L) NI—302.55D8, (M) NI-302.7A8, (N) NI-302.78H12, (O) NI-302.71F6, (P) .11H6, and (Q) NI-302.3D8 do not bind unrelated aggregating protein targets as shown in the binding specificity analysis by direct ELISA Fig. 23: Determination 02 antibody binding epitope by scan ofoverlapping peptides. At the top: pepscan image afier NI-302 antibody hybridization. Below: graphical overviews of peptides sequences and NI—302 antibody binding score to the single peptides are shown. Overlapping amino acids between peptides (putative binding e) being recognized by the NI-302 antibody are highlighted in gray in the consensus sequences. The njugated donkey anti-human IgG Fey detection antibody alone does not bind any linear huntingtin e. (A) NI—302.3 1F11 1 ug/ml on a 21 spot membrane, (B) NI-302.74Cll l ug/ml on a 16 spot membrane, (C) NI- 302.15F9 1 11ng on a 16 spot membrane, (D) NI-302.39G12 1 ug/ml on a 16 spot membrane, (E) NI—302.11A4 l ug/ml on a 16 spot membrane, (F) NI-302.22H9 l ug/ml on a 16 spot membrane, (G) NI-302.44D7 1 ug/ml on a 16 spot membrane, (H) NI-302.37Cl2 l ug/ml on a 16 spot membrane, (1) NI-302.55D8 1 ug/ml on a 16 spot membrane, (J) NI—302.7A8 l ug/ml on a 21 spot membrane, (K) NI-302.78H12 l ug/ml on a 16 spot membrane, (L) NI-302.71F6 1 ug/ml on a 16 spot membrane, (M) NI-302.11H6 l ug/ml on a 21 spot membrane, (N) NI-302.18A1 1 ug/ml on a 21 spot membrane, (0) NI-302.3D8 1 ug/ml on a 21 spot membrane, (P) NI-302.46C9 1 ug/ml on a 21 spot membrane and (Q) NI-302.52C9 l ug/ml on a 21 spot membrane, (R) NI- 302.2A2 1 ug/ml on a 21 spot membrane, (S) NI-302.15E8 1 ug/ml on a 21 spot membrane and (T) NI-302.15D3 1 ug/ml on a 21 spot membrane.
Fig. 24: Immunohistochemical analysis of NI-302 antibodies reveals prominent staining of neuronal intranuclear ions in striatal neurons of late e stage Tg(HDexon1)62pr/1J enic animals at 5 nM (74C11, 39C12, 11A4, 22H9, 78Hl2, 37Cl2, 7D8, 72F10), or SOnM concentrations (15F9, 71F6, 55D8, 44D7, 7A8, 64E5). Mab5492 is a commercially ble N—terminal HTT antibody.
Fig. 25: Basic characterization of R6/l transgenic mouse model Tg(HDexon1)6leb/J. (A) Survival curve, (B) body weight curve and (C) total brain wet weight during the disease progression of this animal model. (D-H) terization of appearance of neuronal intranuclear inclusions with disease progression in the striatum by staining with NI-302.33Cll HTT antibody.
Fig. 26: Basic characterization of B6C3-Tg(HD82Gln)81Dbo/J 82Q) transgenic mouse model. (A) Survival curve, (B) body weight curve during the disease progression and (C) total brain wet weight at end stage of this animal model. (D-F) Characterization of appearance of neuronal intranuclear ions with disease progression in the striatum by staining with Mab5492 HTT antibody.
Fig. 27: Immunohistochemical analysis with 50 nM of NI-302.33C1 1(polyP-epitope) shows staining of neuronal intranuclear inclusions in cortical neurons of four different Huntington e patients (A—D) and in striatal neurons of 270 day old, late disease stage B6.Cg-Tg(HDexon1)6leb/J) enic animals at l (E) and 5 nM (F) tration. No staining is detected in non-transgenic littermates (G), if primary dy is omitted during the staining (H) or if tissue of non-Huntington Disease controls is d with 50 nM 02.33Cl 1.
Fig. 28: Immunohistochemical analysis with 50 nM of NI-302.63F3 (P-rich domain epitope) shows staining ofneuronal intranuclear inclusions (A-C) and ng of some neurites (D) of cortical neurons of four different Huntington Disease ts (A—D) and in striatal neurons of 270 day old, late disease stage B6.Cg-Tg(HDexon1)6leb/J) transgenic animals at l (E) and 50 nM (F) concentration. No staining is detected in non-transgenic littermates (G), if primary antibody is omitted during the staining (H) or if tissue of non-Huntington Disease controls is stained with SOnM ofNI—302.63F3.
Fig. 29: Immunohistochemical analysis with 100 nM of NI-302.35C1 (end Exon 1 epitope) shows staining ofneuronal intranuclear inclusions (A-C) and staining of some neurites (D) of cortical neurons of four different Huntington Disease patients (A-D) and in al neurons of 270 day old, late disease stage B6.Cg-Tg(HDexonl)6leb/J) transgenic s at l (E) and 50 nM (F) concentration. No staining is detected in non-transgenic littermates (G), if primary antibody is omitted during the staining (H) or if tissue of non-Huntington Disease controls is stained with lOOnM ofNI-302.35Cl.
Fig. 30: Immunohistochemical analysis with commercially available anti-polyQ dy Mab1574 (1:2000, Chemicon) shows staining of neuronal intranuclear and cytoplasmic inclusions and staining of some neurites (A, D) of al neurons of four different Huntington e ts (A-D) and in striatal neurons ofpresymptomatic, 150 day old (E) and 270 day old (F), late disease stage B6.Cg-Tg(HDexon1)6leb/J) transgenic animals. No staining is detected in non-transgenic littermates (G), if primary antibody is omitted during the staining (H) or if tissue of non-Huntington e controls is stained with Mab 1574.
Fig. 31: ECso determinations of human-derived HTT antibodies for aggregated HD49 (O), aggregated HD21 (I), soluble GST-HD49 (A) and GST-HD21 (V) Htt Exon 1 proteins using direct ELISA. Some antibodies (e.g. NI-302.64E5 (A) or NI-302.7D8 (B)) seem to have preferred binding to uncut GST-HD49 protein suggesting that these antibodies preferentially recognize uncut soluble GST-HD ucts containing longer polyQ repeats. Antibody NI-302.72F10 (C) shows preference to HD21 constructs and some antibodies (e.g. .4A6 (D), NI-302.12H2 (E) or NI- 302.8Ml (E)) showed high affinity binding with similar EC—Values to all HTT preparation ting that they bind to an epitope that is similar exposed in aggregated and uncut HTT exon 1 constructs in the ELISA assay.
Fig. 32: Characterization of antibody (A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (D) NI-302.4A6, (E) NI-30212H2, (F) NI-302.8Ml and (G) .33Cll (as control) on in vitro HD21, HD35 and HD49 time-resolved in vitro ation reactions by dot-blot (left) and filter retardation assay (right) with preferential binding in particular of NI-302.64E5 and NI-302.72F10 to later (aggregated) reactions of HD35 and/or HD49 in the dot-blot assays and SDS stable aggregates of HD35 and HD49 in the filter ation assay.
Fig. 33: Target specificity analysis by direct ELISA. NI-302 dies (A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (E) NI-302.12H2 and (F) NI-302.8M1 do not bind unrelated aggregating protein s as shown in the g specificity analysis by direct ELISA, except (D) NI-302.4A6 which shows some binding to p53.
Fig. 34: Study of C-terminal domain-binding antibody NI 302.35Cl on behavioral performance during task-specific training and sensorimotor ability in a mice model of HD. (A) The plus—maze analysis was used to investigate the level of anxiety in the R6/l mice. At 6 months of age, NI-302.35Cl treated R6/1 animals spend less time in the open arms, entered the open arms less frequently and did less unprotected head dips on the open arm ed to vehicle treated R6/l animals. Hence the NI- 302.35Cl treated R6/l mice displayed a more anxious phenotype, comparable to the non—transgenic littermates. (B) NI-302.35Cl treated R6/ 1 animal showed an improved performance in the pole test compared to vehicle treated R6/ 1 animals reaching levels similar to non-transgenic animals.
Fig. 35 ination of NI—302 antibody binding epitope by scan of overlapping peptides.
At the top: pepscan image after NI-302 antibody hybridization. Below: graphical ews of es sequences are shown. Overlapping amino acids between peptides (putative binding epitope) being recognized by the NI-302 antibody are shown in the consensus sequence below. The HRP—conjugated donkey anti-human IgG FC’Y ion antibody alone does not bind any linear huntingtin peptide. (A) NI- 302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (D) NI-302.4A6, (E) NI-302.12H2, (F) NI-302.8M1 all antibodies at l ug/ml on the 21 spot membrane.
Fig. 36 Amino acid sequence ent ofthe CDRs in the VH and VL or VK chains ofNI-302 antibodies. Each sequence was checked in terms of ved amino acids, segments, or other motifs revealing an accumulation of tyrosines in the CDRs.
DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to immunotherapy and non-invasive methods for the detection of diseases and/or disorders as well as conditions associated with the presence of pathologic, often mutant and/or aggregated forms of huntingtin (HTT). More cally, the present ion relates to recombinant human-derived monoclonal antibodies and HTT- binding fragments, synthetic and biotechnological derivatives thereof, which have been generated based on ce information obtained from ed human donor populations and are capable of binding to such HTT rns and antigens f. The recombinant human- derived monoclonal antibody of the present invention is advantageously terized by cally binding to mutated and/or aggregated HTT species and/or fragments thereof allowing a targeting for treatment and/or sis of pathological altered HTT species. Due to their human derivation, the resulting recombinant antibodies of the present invention can be reasonably expected to be efficacious and safe as eutic agent, and highly specific as a diagnostic reagent for the detection of pathological HTT without giving false ves.
In addition, the present invention relates to the human monoclonal antibody and any derivatives thereof bed herein for use in the treatment of patients either alone or with other agents utilized for symptoms associated with HTT dosis, wherein the antibody of the t invention and any ofits derivatives is designed to be administered concomitantly with the agent suppressing side effects or sequentially before or after administration of the same. In this context, the anti-HTT antibody and HTT-binding fragment of the present invention are preferably ntially non—immunogenic in human. In one embodiment of the present invention, pharmaceutical compositions are provided comprising both a human monoclonal antibody of the present invention or any derivatives f and one or more drug utilized for symptoms associated with HTT amyloidosis. 1. Definitions Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2.
It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody," is understood to represent one or more antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
Huntingtin (HTT), also known as IT15 is a disease gene linked to Huntington's disease (HD), a egenerative disorder characterized by loss of striatal neurons. It is thought that HD is caused by an expanded, unstable trinucleotide repeat in the HTT gene, which translates as a polyglutamine repeat in the protein product. A fairly broad range in the number of trinucleotide repeats has been identified in normal ls, and repeat numbers in excess of 35-40 have been described as pathological. The HTT locus (NG_009378.1; 4830 to 174286; NCBI RefSquene) is large, spanning 180 kb and consisting of 67 exons.
In this context, it has been demonstrated that an N-terminal fragment of mutant HTT, z'.e. exon 1 protein ofthe HTT gene, with an expanded CAG repeat represents the "toxic" species ofHTT which is sufficient to cause aggregation and a progressive neurological phenotype in enic mice; see, e.g., Mangiarini et al., Cell 87 (1996), 493—506 and Ross et al., Lancet . 10 (2011), 83—98, DiFiglia er al, Science 277 (1997),l990-1993, Gutekunst er al., J Neurosci 19(7) (1999), 2522—2534.
Unless stated otherwise, by "specifically recognizing HTT", "antibody specific to/for HTT" and "anti-HTT dy" antibodies are meant which specifically, generally, and collectively bind to HTT, wherein HTT refer to different forms the HTT including but not limited to the native form ofHTT as well as other forms ofHTT, e.g. pathologically altered HTT, such as d, misfolded and/or aggregated HTT. Provided herein are human-derived antibodies ive for filll-length and/or fragments and/or mutated, misfolded and/or ated forms of HTT.
If not specifically indicated otherwise, the term "HTT", is used interchangeably to specifically refer to the different forms of huntingtin (HTT). The term "HTT" is also used to generally identify other conformers of HTT, for example, pathologically altered forms of HTT such as misfolded and/or ated forms of HTT. Furthermore, unless specifically indicated otherwise the term HTT in particular means HTT exonl and the soluble HTT refers to the ponding GST-fiJsion proteins. The term "HTT" is also used to refer collectively to all types and forms of HTT, such as d HTT. Added letters in front of the terms, e. g. HTT, are used to indicate the organism the particular ortholog is originating from, 6.g. hHTT for human HTT or mHTT for murine origin.
The TT antibodies disclosed herein specifically bind HTT and epitopes thereof and to various conformations of HTT and epitopes thereof. For example, disclosed herein are antibodies that specifically bind pathologically altered HTT species or fragments thereof, such as mutated, misfolded, and/or aggregated forms of HTT or fragments thereof. The term (pathologically) mutated, misfolded, and/or aggregated/aggregates of HTT is used interchangeable to specifically refer to the entioned forms. The term logical) "aggregated forms" or "aggregates" as used herein bes the products of an accumulation or cluster formation due to HTT erroneous/pathological interaction with one another. These aggregates, accumulations or cluster forms may be, ntially consist or consist ofboth HTT and/or HTT fragments and of non-fibrillar oligomers and/or fibrillar ers and fibrils f. As used herein, reference to an antibody tha "specifically binds", "selectively binds", or "preferentially binds" HTT refers to an antibody that does not bind other unrelated ns.
In one example, a HTT antibody disclosed herein can bind HTT or an epitope thereof and Show no binding above about 2 times background for other proteins. In a preferred embodiment, the antibody of the present invention does not substantially recognize unrelated amyloid-forming proteins selected from the group ting of paired l filament tau, TAU, alpha- synuclein, transactive response DNA binding protein 43 (TDP43), islet amyloid polypeptide , transthyrethin (TTR), serum amyloid A (SAA); see es 8, l3, l8 and 31. An antibody that "specifically binds" or "selectively binds" a HTT conformer refers to an antibody that does not bind all conformations of HTT, i.e., does not bind at least one other HTT conformer. For example, disclosed herein are antibodies that can preferentially bind to mutated and/or aggregated forms ofHTT both in vitro and in tissues obtained from patients with diseases and/or ers associated with HTT amyloidosis or with a risk to develop diseases and/or ers ated with HTT amyloidosis. In another embodiment of the present invention the antibodies ofthe present invention specifically targets different regions of the HTT exon 1; see, e.g., Figs. 5, 9, 12, 14, 15. Since the anti-HTT antibodies of the present invention have been isolated from human ts, they may also be called "human auto-antibodies" or "human- derived antibodies" in order to emphasize that those antibodies were indeed expressed initially by the subjects and are not synthetic constructs ted, for example, by means of human immunoglobulin expressing phage libraries or xenogeneic antibodies generated in a transgenic animal expressing part ofthe human immunoglobulin repertoire which hitherto represented one common method for trying to provide human-like antibodies. On the other hand, the human- derived antibody of the present invention may be denoted synthetic, recombinant, and/or biotechnological in order distinguish it from human serum antibodies per se, which may be purified via protein A or affinity column.
However, a particular advantage ofthe therapeutic approach of the present invention lies in the fact that the antibodies of the present invention are derived from B cells or B memory cells from healthy human subjects with no signs of a disease g the occurrence of, or related to misfolded/aggregated HTT and thus are, with a n probability, e ofpreventing a clinically manifest disease related to misfolded/aggregated HTT, or of diminishing the risk of the occurrence of the clinically manifest disease, or of delaying the onset or progression of the ally manifest disease. Typically, the antibodies of the present ion also have already successfully gone through somatic maturation, z'. e. the optimization with respect to selectivity and effectiveness in the high affinity binding to the target HTT molecules by means of somatic variation of the variable regions of the antibody. The knowledge that such cells in Vivo, e.g. in a human, have not been activated by means of related or other logical proteins or cell structures in the sense of an autoimmunological or allergic reaction is also of great l importance since this es a considerably increased chance of successfully living through the clinical test . So to speak, efficiency, acceptability and tolerability have already been trated before the preclinical and clinical development of the prophylactic or eutic antibody in at least one human subject. It can thus be expected that the human anti-HTT antibodies of the present invention, both its target structure-specific efficiency as therapeutic agent and its decreased ility of side effects cantly increase its clinical probability of success.
In contrast, antibodies derived from cDNA library's or phage displays are artificial molecules such as a humanized antibody which is still of murine origin and thus foreign to the human body. Therefore the clinical utility and efficacy of the therapeutic antibodies can be limited by the production of anti-drug antibodies (ADAs), which can influence the efficacy and pharmacokinetics ofthe dies and sometimes lead to serious side effects, see 6.g. Igawa et al. MAbs. 3 (2011), 243-252. In particular, humanized antibodies or antibodies generated with recent human—antibody—generation technologies are in contrast to the human—derived antibodies such as those of the present invention prone to induce an dy response and these human- like antibodies derived from e. g. phage display such as adalimumab have been reported to induce ADA production, see, e.g, Mansour, Br. J. Ophthalmol 91 (2007), 274-276 and Igawa et al., MAbs. 3 (2011), 243-252. Therefore, human-derived antibodies which are not prone to undesired immune response are more beneficial for the patient than artificial les derived from libraries or displays.
The term "peptide" is tood to include the terms "polypeptide" and "protein" (which, at times, may be used interchangeably herein) within its meaning. Similarly, fragments ofproteins and ptides are also contemplated and may be ed to herein as "peptides".
Nevertheless, the term "peptide" preferably denotes an amino acid polymer including at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, more preferably at least contiguous amino acids, still more ably at least 20 contiguous amino acids, and ularly preferred at least 25 contiguous amino acids. In addition, the peptide in accordance with present invention typically has no more than 100 contiguous amino acids, preferably less than 80 contiguous amino acids and more preferably less than 50 contiguous amino acids.
Polzgegtides: As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains oftwo or more amino acids, and does not refer to a c length of the product. Thus, "peptides," "dipeptides," "tripeptides, "oligopeptides, H Hprotein, H Hamino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms.
The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, ation, phosphorylation, amidation and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by turally ing amino acids. A polypeptide may be d from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, ing by chemical synthesis.
A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and ptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a n coupled to at least one carbohydrate moiety that is attached to the protein Via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g., a serine residue or an asparagine residue.
By an "isolated" polypeptide or a nt, variant, or derivative thereof is ed a polypeptide that is not in its natural milieu. No ular level fication is required. For example, an isolated polypeptide can be removed from its native or natural environment.
Recombinantly produced polypeptides and proteins sed in host cells are considered isolated for ed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
"Recombinant peptides, polypeptides or proteins" refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, 1'. e. produced from cells, microbial or mammalian, transformed by an ous recombinant DNA sion construct encoding the fusion protein including the desired peptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Proteins or polypeptides expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.
Included as polypeptides of the present invention are fragments, derivatives, analogs or variants of the foregoing polypeptides and any ations thereof as well. The terms "fragment," nt," "derivative", and "analog" include peptides and polypeptides having an amino acid ce sufficiently r to the amino acid sequence of the natural peptide. The term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common fianctional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the preferred peptides ofthe present invention, in particular to HTT, variants, tives or analogs of either of them. Such variants generally retain the fiinctional activity of the peptides of the t invention. Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or tution(s). These may be naturally occurring variants as well as artificially designed ones.
Furthermore, the terms ent," "variant," "derivative", and "analog" when referring to antibodies or antibody polypeptides of the present invention include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native binding le, antibody, or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of antibodies and antibody polypeptides of the present invention include fragments as described above, and also ptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art- known mutagenesis techniques. Variant polypeptides may comprise conservative or non- conservative amino acid substitutions, deletions or additions. Derivatives of HTT specific binding les, e.g., antibodies and antibody polypeptides of the present invention, are polypeptides which have been altered so as to exhibit onal features not found on the native polypeptide. Examples include fiasion proteins. t polypeptides may also be referred to herein as "polypeptide s". As used herein a "derivative" of a binding molecule or nt f, an antibody, or an antibody polypeptide refers to a subject polypeptide having one or more residues ally derivatized by reaction of a fimctional side group. Also ed as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty rd amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
Determination ofsimilarity and/0r ty ofmolecules: "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide; see Example 35 as well as Fig. 36. An amino acid ofone peptide is r to the corresponding amino acid of a second peptide if it is cal or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M.O., ed., The Atlas of Protein ce and Structure 5, National Biomedical Research Foundation, Washington, DC. (1978), and in Argos, EMBO J. 8 (1989), 779-785.
For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.
"Similarity" between two polynucleotides is determined by comparing the nucleic acid ce of one polynucleotide to the sequence of a polynucleotide. A nucleic acid of one polynucleotide is similar to the corresponding nucleic acid of a second polynucleotide if it is identical or, if the nucleic acid is part of a coding sequence, the tive triplet comprising the nucleic acid encodes for the same amino acid or for a conservative amino acid substitution.
The determination of percent identity or similarity between two sequences is preferably accomplished using the mathematical algorithm ofKarlin and Altschul (1993) Proc. Natl. Acad.
Sci USA 90: 5873-5877. Such an algorithm is incorporated into the BLASTn and BLASTp programs of Altschul et a1. (1990) J. Mol. Biol. 215: 403-410 available at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
The determination of percent identity or similarity is performed with the standard ters of the BLASTn programs for BLAST polynucleotide searches and BLASTp programs for BLAST protein , as recommended on the NCBI webpage and in the "BLAST Program Selection Guide" in t of sequences of a specific length and composition.
BLAST polynucleotide es are performed with the BLASTn m.
For the general ters, the "Max Target Sequences" box may be set to 100, the "Short queries" box may be ticked, the "Expect threshold" box may be set to 1000 and the "Word Size" box may be set to 7 as recommended for short sequences (less than 20 bases) on the NCBI webpage. For longer ces the "Expect threshold" box may be set to 10 and the "Word Size" box may be set to 11. For the scoring parameters the "Match/mismatch Scores" may be set to l,-2 and the "Gap Costs" box may be set to linear. For the Filters and Masking parameters, the "Low complexity regions" box may not be ticked, the "Species-specific repeats" box may not be ticked, the "Mask for lookup table only" box may be ticked, the "DUST Filter Settings" may be ticked and the "Mask lower case letters" box may not be ticked. In general the "Search for short nearly exact matches" may be used in this respect, which provides most of the above indicated settings. Further information in this respect may be found in the "BLAST Program Selection Guide" published on the NCBI webpage.
BLAST protein searches are performed with the BLASTp program. For the l parameters, the "Max Target Sequences" box may be set to 100, the "Short s" box may be ticked, the "Expect threshold" box may be set to 10 and the "Word Size" box may be set to "3". For the scoring parameters the x" box may be set to "BLOSUM62", the "Gap Costs" Box may be set to "Existence: 11 ion: 1", the "Compositional adjustments" box may be set to "Conditional compositional score matrix adjustment". For the Filters and Masking parameters the "Low complexity regions" box may not be ticked, the "Mask for lookup table only" box may not be ticked and the "Mask lower case letters" box may not be .
Modifications of both programs, e.g., in respect of the length of the searched sequences, are performed according to the recommendations in the "BLAST Program Selection Guide" published in a HTML and a PDF version on the NCBI webpage.
Polynucleotides.‘ The term ucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non—conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e. g., DNA or RNA fragments, t in a polynucleotide. By "isolated" nucleic acid or cleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For e, a recombinant polynucleotide encoding an antibody contained in a vector is ered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
Isolated RNA molecules include in vivo or in vitro RNA ripts of polynucleotides of the t invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules ed synthetically. In addition, polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome g site, or a transcription terminator.
As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part ofa coding region, but any flanking ces, for example promoters, ribosome binding sites, transcriptional ators, s, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) s. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g, a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or c acid of the invention may encode heterologous coding regions, either filS€d or d to a nucleic acid encoding a binding molecule, an antibody, or fragment, variant, or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operable associated with one or more coding regions. An operable ation is when a coding region for a gene product, e.g, a polypeptide, is associated with one or more regulatory sequences in such a way as to place sion of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a ptide coding region and a promoter associated therewith) are "operable associated" or "operable linked" if ion of promoter fimction results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage n the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operable associated with a nucleic acid encoding a ptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a pecific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control ts, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operable associated with the polynucleotide to direct cell-specific ription. Suitable promoters and other transcription control s are disclosed herein.
A y of transcription control regions are known to those skilled in the art. These e, without limitation, transcription l regions which fimction in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron—A), simian Virus 40 (the early promoter), and retroviruses (such as Rous sarcoma Virus). Other transcription control regions include those d from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other ces capable ofcontrolling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g. inducible by interferons or interleukins). , promoters Similarly, a variety of translation l elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
In other ments, a polynucleotide of the present invention is RNA, for example, in the form of ger RNA (mRNA).
Polynucleotide and nucleic acid coding s of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal esis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N—terminus ofthe polypeptide, which is cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In n embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a fimctional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is le associated with it. Alternatively, a heterologous ian signal peptide, or a fimctional derivative thereof, may be used. For example, the wild-type leader sequence may be tuted with the leader sequence of human tissue plasminogen activator (TPA) or mouse B-glucuronidase.
A "binding molecule" as used in the context of the present invention relates primarily to antibodies, and fragments thereof, but may also refer to other tibody molecules that bind to HTT including but not limited to hormones, receptors, ligands, ankyrins, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs) as well as cell-cell adhesion molecules such as members of the cadherin, intergrin, C-type lectin and immunoglobulin (Ig) amilies. Thus, for the sake of clarity only and without restricting the scope of the present invention most of the following embodiments are discussed with respect to antibodies and antibody-like molecules which represent the preferred binding les for the development of therapeutic and diagnostic agents.
Antibodies: The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a binding molecule which comprises at least the le domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.
Basic immunoglobulin structures in vertebrate systems are vely well understood; see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
As will be discussed in more detail below, the term "immunoglobulin" comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, u, CL, 8, 8) with some subclasses among them (e.g, yl-y4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g, IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional specialization. d versions of each of these classes and isotypes are readily discernible to the skilled n in view of the instant disclosure and, accordingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following sion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two cal light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides ofmolecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration n the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable .
Light chains are classified as either kappa or lambda (K, k). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the globulins are generated either by hybridomas, B cells or cally engineered host cells. In the heavy chain, the amino acid sequences run from an inus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
Both the light and heavy chains are divided into regions of structural and functional homology.
The terms "constan " and "variable" are used fimctionally. In this regard, it will be appreciated that the variable s ofboth the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. sely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental ty, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region s increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N—terminal portion is a variable region and at the C—terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively. As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the le region that defines a three dimensional n-binding site. This quaternary antibody structure forms the antigen- binding site present at the end of each arm of the Y. More specifically, the antigen—binding site is defined by three CDRs on each of the VH and VL chains. Any antibody or immunoglobulin fragment which contains sufficient structure to specifically bind to HTT is denoted herein interchangeably as a ng nt" or an "immunospecific fragmen ." In lly occurring antibodies, an antibody comprises six hypervariable regions, sometimes called "complementarity determining regions" or "CDRs" present in each antigen-binding domain, which are short, non-contiguous sequences of amino acids that are specifically positioned to form the n-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" which show less inter-molecular variability. The framework regions y adopt a B-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the B-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter—chain, valent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This mentary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined; see, "Sequences of Proteins of logical Interest," Kabat, E., et al., US. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol.
Biol, 196 (1987), 901-917, which are incorporated herein by nce in their entireties.
In the case where there are two or more ions of a term which is used and/or ed within the art, the definition ofthe term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term ementarity determining region" ("CDR") to describe the ntiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides.
This particular region has been described by Kabat et al., US. Dept. of Health and Human Services, "Sequences of ns of Immunological Interest" (1983) and by Chothia and Lesk, J. Mol. Biol, 196 (1987), 901-917, which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid es when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table I as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular ariable region or CDR of the human IgG subtype of antibody given the variable region amino acid sequence of the antibody.
Table I: CDR Definitions1 Kabat a VH CDRI 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR] 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96 1Numbering of all CDR definitions in Table I is according to the numbering conventions set forth by Kabat et al. (see below).
Kabat et al. also defined a numbering system for le domain sequences that is applicable to any antibody. One ofordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any le domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise ed, references to the numbering of specific amino acid residue positions in an antibody or antigen-binding fragment, variant, or derivative thereof of the present invention are according to the Kabat numbering system, which however is theoretical and may not y apply to every antibody of the present invention.
For e, depending on the on of the first CDR the following CDRs might be shifted in either direction.
Unless human-derived onal antibodies or an antigen-binding fragment, synthetic or biotechnological derivative thereof as particularly preferred embodiments of the present are referred to, antibodies or antigen-binding fragments, immunospecific fragments, ts, or tives thereof of the invention include, but are not limited to, onal, monoclonal, pecific, human, humanized, primatized, murinized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs , single-chain antibodies, disulfide-linked Fvs (dev), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g, anti-Id antibodies to antibodies disclosed herein). ScFV molecules are known in the art and are described, e. g., in US patent 5,892,019. Immunoglobulin or antibody les of the invention can be of any type (e. g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
In one embodiment, the antibody of the present invention is not IgM or a derivative thereof with a pentavalent structure. Particular, in specific applications of the present invention, especially therapeutic use, Ing are less useful than IgG and other bivalent antibodies or corresponding binding molecules since Ing due to their pentavalent structure and lack of affinity maturation often show unspecific cross-reactivities and very low affinity.
In a particularly preferred embodiment, the antibody of the present invention is not a polyclonal antibody, 1'. e. it substantially consists of one particular antibody species rather than being a mixture obtained from a plasma immunoglobulin .
Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the ty or a portion of the ing: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are HTT g fragments which comprise any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains.
Antibodies or immunospecific fragments thereof of the t invention may be from any animal origin including birds and s. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another ment, the variable region may be condricthoid in origin (e.g. , from sharks).
In one , the antibody of the present invention is a human-derived monoclonal dy isolated from a human, wherein the B cell sing the antibody is isolated from a human and in turn the dy or preferably the cDNA encoding the variable domain and optionally the cDNA for the cognate constant domain. Optionally, the framework region of the human antibody is aligned and adopted in accordance with the pertinent human germ line le region sequences in the database; see, e.g, Vbase (https://vbase.mrc-cpe.cam.ac.uk/) (https://www.vbase2.org/) hosted by the MRC Centre for Protein Engineering (Cambridge, UK).
For example, amino acids considered to potentially deviate from the true germ line sequence could be due to the PCR primer sequences incorporated during the cloning s. Compared to artificially ted human-like antibodies such as single chain antibody fragments (scFvs) from a phage displayed antibody library or xenogeneic mice the human monoclonal antibody of the present invention is characterized by (i) being obtained using the human immune response rather than that of animal surrogates, z'.e. the antibody has been generated in response to natural HTT in its relevant conformation in the human body, (ii) having protected the individual or is at least significant for the presence of HTT, and (iii) since the antibody is of human origin the risks of reactivity against self—antigens is minimized. Thus, in accordance with the present invention the terms "human monoclonal antibody", "human monoclonal autoantibody", "human antibody" and the like are used to denote a HTT binding molecule which is of human origin, Le. which has been isolated from a human cell such as a B cell or hybridoma thereof or the cDNA of which has been directly cloned from mRNA of a human cell, for example a human memory B cell. A human antibody is still "human", z'.e. human-derived even if amino acid substitutions are made in the antibody, e.g., to improve binding characteristics. In this context, contrary to humanized antibodies and otherwise human- like antibodies, see also the discussion infra, the human-derived antibodies of the present invention are characterized by comprising CDRs which have been seen by human body and ore are substantially devoid of the risk of being immunogenic. Therefore, the antibody of the t invention may still be denoted human—derived if at least one, preferably two and most preferably all three CDRs of one or both the variable light and heavy chain ofthe antibody are derived from the human antibodies illustrated herein.
In one embodiment the human-derived antibodies of the present invention comprises heterologous regions compared to the natural occurring antibodies, 6.g. amino acid substitutions in the framework region, nt region exogenously quCd to the variable region, different amino acids at the C— or N- terminal ends and the like.
Antibodies derived from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not s endogenous immunoglobulins, as described infra and, for example in, US patent no 5,939,598 by Kucherlapati et a]. are denoted human-like antibodies in order distinguish them from truly human antibodies of the present invention.
For example, the paring of heavy and light chains of human-like antibodies such as tic and semi-synthetic antibodies typically isolated from phage display do not necessarily reflect the original paring as it occurred in the original human B cell. Accordingly Fab and scFv fragments obtained from recombinant expression ies as commonly used in the prior art can be ered as being artificial with all possible associated effects on immunogenicity and stability.
In contrast, the present invention es ed affinity-matured antibodies from ed human subjects, which are characterized by their therapeutic utility and their tolerance in man.
As used herein, the term "rodentized antibody" or "rodentized immunoglobulin" refers to an antibody comprising one or more CDRs from a human antibody of the t invention; and a human framework region that ns amino acid substitutions and/or deletions and/or insertions that are based on a rodent antibody sequence. When referred to rodents, preferably sequences originating in mice and rats are used, wherein the antibodies comprising such sequences are referred to as ized" or "ratinized" respectively. The human immunoglobulin providing the CDRs is called the "paren " or "acceptor" and the rodent antibody providing the framework changes is called the "donor". Constant regions need not be present, but if they are, they are usually substantially identical to the rodent antibody constant regions, 1'. e. at least about 85 % to 90 %, preferably about 95 % or more identical. Hence, in some embodiments, a lull-length murinized human heavy or light chain immunoglobulin contains a mouse constant region, human CDRs, and a substantially human framework that has a number of "murinizing" amino acid substitutions. Typically, a ized antibody" is an antibody comprising a zed variable light chain and/or a murinized variable heavy chain.
For example, a murinized antibody would not encompass a typical chimeric antibody, e.g., e the entire variable region of a chimeric dy is non-mouse. A modified antibody that has been "murinized" by the process of "murinization" binds to the same n as the parent antibody that es the CDRs and is usually less immunogenic in mice, as compared to the parent antibody. The above explanations in respect of "murinized" antibodies apply analogously for "rodentized" antibodies, such as "ratinized antibodies", n rat sequences are used instead of the .
As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge ) domain, a CH2 domain, a CH3 domain, or a variant or fragment f. For example, a binding polypeptide for use in the invention may se a polypeptide chain comprising a CHl ; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHl domain, at least a n of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Further, a binding polypeptide for use in the invention may lack at least a n of a CH2 domain (e. g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
In certain dies, or antigen-binding nts, variants, or derivatives thereof disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain n-containing monomers of the invention are not cal. For example, each monomer may comprise a different target binding site, forming, for example, a bispecific antibody or y.
As used herein, the term "bispecific" or "bifimctional" antibody molecule is an antibody molecule that has two different e/antigen binding sites, and accordingly has binding specificities for two ent target epitopes. These two epitopes may be epitopes of the same antigen or erent antigens. In contrast o a "bivalent antibody" may have binding sites ofidentical antigenic specificity. Methods of making a bispecific antibody are known in the art, 6. g. chemical conjugation of two different monoclonal antibodies as illustrated in Example 36 or for example, also chemical conjugation of two antibody fragments, for e, of two Fab fragments (Brennan et al., Science 229 (1985), 81-83; Nitta et al., Eur. J. Immunol. 19 , 1437—1441; Glennie et al., J. Immunol. 139 (1987), 2367-2375; Jung et al., Eur. J. Immunol., 21 , 2431-2435). Alternatively, bispecific antibodies are made recombinantly (Gruber et al., J. Immunol. 152 (1994), 5368—5374; Kurucz et al., J. Immunol. 154 (1995), 4576-4582; Mallender and Voss, J. Biol. Chem. 269 (1994), 199-206). Traditionally, the recombinant production of bispecific dies is based on the co-expression oftwo immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different binding specificities. Because ofthe random assortment ofheavy and light chains, a potential mixture often different antibody structures are produced of which only one has the desired binding specificity (Milstein and Cuello, Nature 305 (1983), 537-540; Lanzavecchia and Scheidegger, Eur. J. Immunol. 17 (1987), 105—111. An alternative approach involves fusing the variable domains with the d binding specificities to heavy chain constant region including at least part of the hinge region, CH2 and CH3 regions. In one embodiment the CH1 region ning the site necessary for light chain binding is present in at least one of the fusions. DNA encoding these s, and if desired the light chains are inserted into separate expression vectors and are then co-transfected into a suitable host organism. It is possible though to insert the coding sequences for two or all three chains into one expression vector.
In another embodiment, the antibodies, or antigen-binding fragments, ts, or derivatives thereof disclosed herein are composed of a single polypeptide chain such as scFvs and are to be sed ellularly (intrabodies) for potential in viva therapeutic and diagnostic applications.
The heavy chain ns of a binding polypeptide for use in the diagnostic and treatment methods sed herein may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain n can se a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 le.
As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain. Preferably, the light chain portion comprises at least one of a VL or CL domain.
The minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids.
Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same e chain. In the t invention, a peptide or polypeptide epitope recognized by antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of HTT, in particular of a N- terminal, polyP region, P-rich region or the C-terminal region of exon 1.
By "specifically binding", or "specifically recognizing", used interchangeably herein, it is generally meant that a g molecule, e.g., an antibody binds to an e Via its antigen- g domain, and that the binding entails some complementarity n the antigenbinding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, Via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to y the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher city than it has for related epitope "D".
Where t, the term "immunological binding characteristics," or other binding characteristics of an antibody with an antigen, in all of its grammatical forms, refers to the specificity, affinity, cross—reactivity, and other binding characteristics of an antibody.
By "preferentially binding", it is meant that the binding molecule, e.g., antibody specifically binds to an epitope more y than it would bind to a d, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given e would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related e.
By way of non-limiting example, a binding molecule, e.g, an antibody may be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (KD) that is less than the antibody's KD for the second epitope. In another non-limiting example, an dy may be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's KD for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's KD for the second epitope.
In another non-limiting e, a binding molecule, e.g, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope. In another miting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of ude less than the antibody's k(off) for the second epitope.
A binding molecule, e.g., an antibody or antigen—binding fragment, variant, or derivative sed herein may be said to bind HTT or a fragment, variant or specific conformation thereof with an off rate (k(off)) of less than or equal to 5 x 10'2 sec'l, 10'2 sec'l, 5 x 10'3 sec‘1 or '3 sec'l. More ably, an antibody ofthe invention may be said to bind HTT or a fragment, variant or specific conformation thereof with an off rate (k(off)) less than or equal to 5 x 10'4 sec'l, 10'4 sec'l, 5 x 10'5 sec'l, or 10‘5 sec"1 5 x 10'6 sec'l, 10'6 sec'l, 5 x 10'7 sec"1 or 10'7 sec'l.
A binding molecule, e.g., an dy or n-binding fragment, variant, or derivative disclosed herein may be said to bind HTT or a fragment, variant or specific conformation thereof with an on rate (k(on)) of greater than or equal to 103 M"1 sec'l, 5 X 103 M"1 sec], 104 M"1 sec"1 or 5 X 104 M"1 sec‘l. More preferably, an antibody of the invention may be said to bind HTT or a fragment, t or specific conformation thereofwith an on rate (k(on)) greater than or equal to 105 M"1 sec'l, 5 x 105 M‘1 sec'l, 106 M"1 sec'l, or 5 x 106 M"1 sec"1 or 107 M"1 sec'l.
A g molecule, e.g., an dy is said to competitively inhibit g of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for e, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
As used herein, the term ty" refers to a measure of the strength of the binding of an individual epitope with the CDR of a binding molecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et (11., dies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) at pages 27—28. As used herein, the term "avidity" refers to the overall stability ofthe x between a population of immunoglobulins and an antigen, that is, the fiinctional combining strength of an immunoglobulin mixture with the antigen; see, e.g., Harlow at pages 29-34. Avidity is d to both the y of individual immunoglobulin molecules in the population with specific epitopes, and also the valences of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high y. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. n and Company New York, N Y (1992), and methods described herein. General techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody- antigen ction can vary if measured under different conditions, 6.g. , salt concentration, pH.
Thus, ements of affinity and other antigen-binding parameters, e.g., K13, ICso, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.
Binding les, e.g., antibodies or n-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody, specific for one antigen, to react with a second n; a measure ofrelatedness between two different antigenic substances. Thus, an antibody is cross reactive if it binds to an epitope other than the one that induced its ion. The cross reactive epitope generally ns many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.
For e, certain antibodies have some degree of cross—reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An dy may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that e.
Binding les, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their binding affinity to HTT and/or d, misfolded, and/or aggregated HTT species and/or fragments thereof.
Preferred binding affinities include those with a dissociation constant or Kd less than 5 x 10'2 M, 10'2 M, 5 x 10'3 M, 10'3 M, 5 X 10'4 M, 10'4 M, 5 x 10'5 M, 10'5 M, 5 X 10'6 M, 10'6 M, 5 x10' 7M, 10'7M, 5 X 10'8M, 10'8M, 5 x 10'9M, 10'9 M, 5 X lO'lOM, 10'") M, 5 x 10'11 M, 10'11 M, 5 x 10‘12M, , 5 X 10'13 M, 10‘13M, 5 x10'14M,10'14M,5 x 10‘15M, or 10'15M.
As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the s immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CH1 domain" includes the first most amino terminal constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
As used herein the term "CH2 domain" includes the portion of a heavy chain molecule that extends, e.g, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering ; see Kabat EA et a]. op. cit). The CH2 domain is unique in that it is not closely paired with another . Rather, two N—linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and ses imately 108 residues.
As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 . This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N—terminal antigen—binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, , and lower hinge s; see Roux et al., J. Immunol. 161 (1998), 4083-4090.
As used herein the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two de bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
As used herein, the terms d", "fused" or "filSlOIl" are used interchangeably. These terms refer to the g together oftwo more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame filSlOIl" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant filSlOIl protein is a single protein containing two or more segments that pond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). Although the reading frame is thus made continuous throughout the filSCd segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides ng the CDRs of an immunoglobulin variable region may be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "filsed" CDRS are co-translated as part of a continuous polypeptide.
The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process es any manifestation of the functional ce of the gene within the cell including, without limitation, gene knockdown as well as both ent expression and stable expression. It es without limitation transcription ofthe gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA ), small interfering RNA ) or any other RNA product, and the translation of mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., enylation, or ptides with post translational modifications, e.g, methylation, glycosylation, the addition of lipids, ation with other protein subunits, lytic cleavage, and the like.
As used herein, the term "sample" refers to any biological material obtained from a t or patient. In one aspect, a sample can comprise blood, peritoneal fluid, CSF, saliva or urine. In other aspects, a sample can comprise whole blood, blood plasma, blood serum, B cells enriched from blood samples, and cultured cells (e.g., B cells from a subject). A sample can also include a biopsy or tissue sample including neural . In still other aspects, a sample can comprise whole cells and/or a lysate of the cells. Blood samples can be collected by methods known in the art. In one aspect, the pellet can be resuspended by vortexing at 4°C in 200 ul buffer (20 mM Tris, pH. 7.5, 0.5 % Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma Protease Inhibitor, and IX Sigma Phosphatase Inhibitors l and 2). The suspension can be kept on ice for min. with intermittent vortexing. After spinning at 15,000 x g for 5 min at about 4°C, aliquots of supernatant can be stored at about -70°C.
Diseases: Unless stated otherwise, the terms "disorder" and "disease" are used interchangeably herein and se any undesired physiological change in a subject, an animal, an isolated organ, tissue or cell/cell culture.
Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative er terized by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene (Huntington's Disease Collaborative Research Group, Cell 72(6) (1993), 971—983), wherein the pathogenic threshold of this expansion is approximately 37 repeats, whereas fewer repeats below this number do not result in pathogenesis, see e. g. Trottier et al., Nature 378(6555) (1995), 403—406. The expansion of the CAG trinucleotide repeat results in an ed polyglutamine (Poly-Gln, Poly-Q) tract in the amino terminus of the huntingtin protein (HTT), which is associated with the aggregation ofHTT. However, the precise mechanism leading to the accumulation ofHTT and its associated symptoms has not been elucidated so far.
Studies have shown that both flanking regions of the polyglutamine Q) tract, z'.e. amino- al region consisting of an amphipathic alpha-helical targeting domain and the carboxy— terminal region characterized by two proline tracts (Poly-P region) and a leucine—proline—rich tract (P-rich region) seem to be critical in mediating the toxicity of the mutated HTT, see 6.g.
Caron et al., PNAS 110 (2013), 14610—14615.
The mechanism contributing to the pathological symptoms of HD, such as hyperkinesia, hypokinesia, mental and movement disorders ing disturbances of affect and the drive, lack of motor persistence, thoughtless and impulsive behavior, resignation and sion, disorders of visual information processing, subcortical dementia, loss of ive ies, disorientation and paucity of speech, delusions, restlessness of the arms, legs, face, head and the trunk, choreic hyperkinesis, dysarthria, dysphagia, anarthria, dystonias, has not been elucidated so far. Possible mechanisms e but are not limited to a reduced flexibility of the hinge region due to the expanded Poly-Q tract of HTT as well as to proteases which lead to the formation of different HTT fragments due to the expanded Poly-Q tract.
Since the antibodies of the present invention have been shown to be eutically effective in a HD mouse model, see e.g. Example 24 and Fig. 17 as well as Example 34 and Fig. 34 and in addition are capable of binding to HTT amyloids in tissue sections from HD patients, see e.g.
Example 31 and Fig. 27-30, the human-derived antibodies and biotechnological derivatives thereof are useful in both the ent and diagnosis of HD and the above-mentioned symptoms. Therefore, in one embodiment of the present invention the dies of the present invention, binding molecules having substantially the same binding specificities of any one thereof, the polynucleotides, the vectors or the cells of the present invention are used for preparation of a pharmaceutical or stic composition for prophylactic and/or therapeutic treatment of HD, in particular HTT amyloidosis es and/or disorders, for monitoring disease progression and/or treatment response, and for the diagnosis of diseases associated with HTT amyloidosis.
Treatment.‘ As used herein, the terms "treat" or "treatment" refer to both therapeutic ent and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of cardiac deficiency.
Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, shment of extent of disease, stabilized (z'.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or , whether detectable or undetectable. "Treatment" can also mean prolonging al as ed to expected al if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the manifestation of the condition or disorder is to be prevented.
If not stated otherwise the term "drug, medicine," or "medicament" are used interchangeably herein and shall include but are not limited to all (A) articles, medicines and preparations for internal or external use, and any substance or mixture of substances intended to be used for diagnosis, cure, mitigation, treatment, or prevention of disease of either man or other animals; and (B) articles, nes and ations (other than food) ed to affect the structure or any fimction of the body of man or other animals; and (C) articles intended for use as a component of any e specified in clause (A) and (B). The term "drug, medicine," or "medicament" shall include the complete formula of the preparation intended for use in either man or other animals containing one or more "agents, compounds", ances" or "(chemical) compositions" as and in some other context also other pharmaceutically inactive ents as fillers, disintegrants, lubricants, glidants, binders or ensuring easy transport, disintegration, disaggregation, dissolution and biological availability ofthe "drug, medicine," " at or amen an intended target location within the body of man or other animals, e.g, at the skin, in the h or the intestine. The terms "agent," "compound", or "substance" are used interchangeably herein and shall include, in a more particular context, but are not limited to all pharmacologically active agents, z‘.e. agents that induce a desired biological or pharmacological effect or are investigated or tested for the capability of inducing such a possible pharmacological effect by the methods of the present invention.
By "subject" or "individual" or l" or "patien " or "mammal", is meant any subject, ularly a mammalian subject, e.g., a human patient, for whom diagnosis, prognosis, prevention, or y is desired.
Pharmaceutical carriers: ceutically acceptable rs and administration routes can be taken from corresponding literature known to the person skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0306472, Vaccine Protocols 2nd Edition by Robinson et al., Humana Press, , New , USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery s. 2nd Edition by Taylor and Francis. (2006), ISBN: 01630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline ons, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be ated by well-known conventional s. These pharmaceutical compositions can be administered to the subject at a suitable dose.
Administration of the suitable compositions may be effected by different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, enous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous nes. Pharmaceutical itions for oral administration, such as single domain antibody molecules (e.g., "nanobodiesTM") etc. are also ged in the present invention. Such oral formulations may be in tablet, capsule, powder, liquid or o lid form. A tablet may comprise a solid carrier, such as gelatin or an adjuvant.
Formulations for rectal or vaginal administration may be presented as a itory with a suitable carrier; see also O'Hagan er al., Nature Reviews, Drug Discovery 2(9) (2003), 727- 735. Further guidance regarding formulations that are le for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985) and corresponding updates. For a briefreview of methods for drug delivery see Langer, Science 249 (1990), 1527-1533. 11. Antibodies of the present ion The present ion generally relates to human-derived anti-HTT antibodies and HTT- binding fragments thereof, which preferably demonstrate the immunological binding characteristics and/or biological properties as ed for the antibodies illustrated in the Examples. In accordance with the present invention human monoclonal antibodies specific for HTT were cloned from B cells of a pool of healthy human subjects. However, in another embodiment of the t invention, the human monoclonal anti—HTT antibodies might also be cloned from B cells of patients showing symptoms of a disease and/or disorder associated with HTT amyloidosis.
In the course of the experiments performed in accordance with the present invention, antibodies present in the conditioned media of cultured human memory B cell were evaluated for their ty to bind to HTT and to more than 10 other proteins including bovine serum albumin (BSA); see Examples 8, 13, 18, 31 and 33. Only the B-cell supematants able to bind to the HTT protein but not to any of the other ns in the screen were selected for firrther analysis, including determination of the antibody class and light chain subclass. The selected B-cells were then processed for antibody cloning.
In brief, this consisted in the tion of messenger RNAs from the selected B—cells, retro- transcription by RT-PCR, amplification of the antibody-coding regions by PCR, cloning into plasmid vectors and sequencing. ed human antibodies were then produced by recombinant expression in HEK293 or CHO cells and purification, and subsequently characterized for their capacity to bind human HTT protein. The ation of various tests, e. g. recombinant expression of the dies in HEK293 or CHO cells and the uent characterization of their binding specificities towards human HTT protein, and their distinctive binding to ogically mutated and/or aggregated forms thereof confirmed that for the first time human antibodies have been cloned that are highly specific for HTT and distinctively recognize and ively bind the ogically aggregated forms of HTT protein. In some cases, mouse chimeric antibodies were also generated on the basis of the variable domains of the human antibodies of the present invention.
Thus, the present invention generally relates to recombinant human-derived monoclonal anti- HTT dies and HTT-binding fragments, tic and biotechnological derivatives and variants thereof. In one embodiment of the invention, the antibody is capable of binding human HTT.
In one embodiment of the present invention the antibody cally binds an epitope in a polyP-region of HTT, which comprises the amino acid sequence PPPPPPPP (NI-302.33C11; NI-302.44D7; NI-302.7A8; NI-302.3D8; NI-302.46C9) (SEQ ID Nos. 139, 151, 154, 158, 161), amino acid ce PPPPPP (NI-302.11H6, NI-302.18A1, NI-302.52C9 (SEQ ID Nos.: 157, 159, 160), amino acid ce PPPPPPPPPPP (NI-302.74C11, NI-302.15F9, NI- 302.39G12, NI-302.11A4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI- 302.71F6 (SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156), an epitope in the P—rich— region which comprises the amino acid ce PQPPPQAQPL (NI-302.63F3 SEQ ID No. 140, NI-302.64E5 SEQ ID No. 200), the amino acid sequence PPPQLPQPPP (NI-302.31F11, SEQ ID No. 141), the amino acid sequence QAQPLLPQPQPPPPP (NI-302.2A2; SEQ ID No. 142), or the amino acid sequence PPPQLPQPPPQAQPL (NI302.15D3; SEQ ID No. 143), an e in the C-terminal region which comprise the amino acid sequence AEEPLHRP (NI-302.35C1, SEQ ID No. 145) or PPPGPAVAEEPLH (NI- 302.72F10, SEQ ID No. 202), an epitope in the N-terminal region which comprises the amino acid sequence KAFESLKSFQ (NI-NI-302.15E8, SEQ ID No. 144) or an epitope in the P/Q- egion which comprises the amino acid sequence QQQPPP (NI-302.7D8 SEQ ID No. 201), or a conformational epitope.
In another embodiment, the present invention is directed to an anti—HTT antibody, or antigen- binding fragment, t or biotechnological derivatives thereof, where the antibody specifically binds to the same epitope in a polyP-region ofHTT as a reference antibody selected from the group ting ofNI-302.33C11, NI-302.74C11, NI-302.15F9, NI-302.39G12, NI- 302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, 302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI- 302.52C9, NI-302.46C9. Epitope mapping identified a sequence within the region of human HTT including amino acids PPPPPPPPPPP (SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156) as the unique linear epitope recognized by antibodies NI-302.74C1l, NI- 302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI- H12, NI-302.71F6 ofthis invention. Additionally, epitope mapping fied a sequence within the polyP-region of human HTT including amino acids PPPPPPPP (SEQ ID Nos. 139, 151, 154, 158, 161) as the unique linear epitope recognized by antibodies NI-302.33C11, NI- 302.44D7, NI—302.7A8, NI—302.3D8, NI-302.46C9 ofthis invention, and amino acids PPPPPP (SEQ ID Nos: 157, 159, 160) as the unique linear epitope recognized by antibodies NI- 302.11H6, NI-302.18A1, NI-302.52C9 of this invention. Therefore, in one embodiment the antibody of the present ion is provided, wherein the antibody specifically binds to an epitope in a polyP—region of HTT, which comprises the amino acid sequence PPPPPPPPPPP (SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156), PPPPPPPP (SEQ ID Nos. 139, 151, 154, 158, 161), or PPPPPP (SEQ ID Nos: 157, 159, 160).
In one ment, the present invention is directed to an TT antibody, or antigen- binding fragment, variant or biotechnological derivatives f, where the antibody specifically binds to the same epitope in the P-rich region of HTT as a reference antibody selected from the group consisting of NI-302.63F3, NI-302.31F11, NI-302.2A2, and NI- 302.15D3. Epitope mapping identified a sequence within the P-rich-region of human HTT including amino acids AQPL (SEQ ID No. 140) as the unique linear epitope recognized by antibody NI-302.63F3 of this invention, PPPQLPQPPP (SEQ ID No. 141), as the unique linear epitope recognized by antibody NI-302.31F11 of this invention, PPPQLPQPPP (SEQ ID No. 141), as the unique linear epitope recognized by antibody NI- 302.31F11 of this invention, QAQPLLPQPQPPPPP (SEQ ID No. 142) as the unique linear epitope ized by antibody .2A2, PPPQLPQPPPQAQPL (SEQ ID No. 143) as the unique linear epitope recognized by antibody NI302. 15D3, PQPPPQAQPL as the unique linear epitope recognized by antibody NI302.64E5. ore, in one embodiment the antibody ofthe present ion is provided, wherein the antibody specifically binds to an epitope in the P- rich—region of HTT which comprises the amino acid sequence AQPL (SEQ ID No. 140), PPPQLPQPPP (SEQ ID No. 141), QAQPLLPQPQPPPPP (SEQ ID No. 142), PPPQLPQPPPQAQPL (SEQ ID No. 143).
In another embodiment the present invention is directed to an anti-HTT dy, or antigen- binding fragment, variant or biotechnological derivatives thereof, wherein the antibody specifically binds to the same epitope in the polyQ/polyP—region ofHTT as reference antibody NI-302.7D8. Epitope mapping identified a sequence within the Q/P-rich—region ofhuman HTT including amino acids QQQQQQQPPP (SEQ ID No. 201) as the unique linear epitope recognized by dy NI-302.7D8 of this invention. ore, in one embodiment the antibody of the present invention is provided, wherein the antibody cally binds to an epitope in in the polyQ/polyP-region of HTT which comprises the amino acid sequence QQQQQQQPPP (SEQ ID No. 201) In one embodiment, the present invention is directed to an anti—HTT antibody, or antigen- binding fragment, variant or biotechnological derivatives thereof, where the antibody specifically binds to the same e in the C-terminal region ofHTT as a reference antibody selected from the group consisting of NI-302.35C1. Epitope mapping identified a sequence within the C—terminal region ofhuman HTT including amino acids PPGPAVAEEPLHRP (SEQ ID No. 145) as the unique linear epitope ized by antibody .35Cl ofthis invention.
Therefore, in one embodiment the antibody of the present invention is provided, wherein the antibody cally binds to an epitope in in the C-terminal region of HTT which comprises the amino acid sequence PPGPAVAEEPLHRP (SEQ ID No. 145).
In a fiirther embodiment, the present invention is directed to an TT antibody, or antigen- binding nt, variant or biotechnological derivatives thereof, where the antibody specifically binds to the same epitope in the C-terminal region of HTT as reference antibody NI-302.72F10. Epitope mapping identified a ce within the C-terminal region of human HTT including amino acids PPPGPAVAEEPLH (SEQ ID No. 202) as the unique linear epitope ized by antibody NI-302.72F10 of this ion. Therefore, in one embodiment the dy of the present invention is provided, wherein the antibody specifically binds to an epitope in in the C-terminal region of HTT which comprises the amino acid sequence VAEEPLH (SEQ ID No. 202).
In another embodiment, the present invention is directed to an anti-HTT antibody, or antigen- binding fragment, variant or biotechnological derivatives thereof, where the antibody cally binds to the same epitope in the N—terminal region of HTT as reference antibody NI-302.15E8. Epitope mapping identified a ce within the N-terminal region of human HTT including amino acids KAFESLKSFQ (SEQ ID No. 144) as the unique linear epitope recognized by antibody NI-302.15E8 of this invention. Therefore, in one embodiment the antibody of the present invention is provided, wherein the antibody specifically binds to an epitope in in the N-terminal region of HTT which comprises the amino acid ce KAFESLKSFQ (SEQ ID No. 144).
In one embodiment, the present invention is directed to an anti-HTT antibody, or n- binding fragment, variant or biotechnological derivatives thereof, where the antibody specifically binds to the same e ofHTT exon 1 as a reference antibody selected from the group consisting of NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1 which have been shown not to bind to linear peptides ofHTT exon 1 but ated HTT exon 1 proteins with 21 or 49 polyQ (HD21 and HD49) with high affinity and an ECso value in the subnanomolar range; see, e. g., Example 25 and Figure 20 for ew. ore, in one preferred embodiment the antibody ofthe present invention specifically binds aggregated forms of HTT, in particular protein aggregates derived from HTT exon 1 with an ECso value of below 1 nM, preferably below 0,1 nM and most ably below 0,01 nM.
Furthermore, without intending to be bound by initial experimental ations as demonstrated in the Examples and shown in Figures, the human monoclonal NI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31F11, NI-302.6N9, NI-302.46C9, NI-302.8F1, NI- 302.74C11, NI-302.15F9, .39Gl2, .11A4, NI-302.22H9, NI-302.44D7, NI- 302.55D8, NI—302.7A8, NI—302.78Hl2, NI—302.71F6, NI-302.11H6, NI-302.3D8, and NI.302- 64E5 and NI.302-72F10 anti-HTT antibodies of the present invention are preferably characterized in specifically binding to pathological mutated and/or aggregated HTT and to substantially smaller affinity recognizing HTT in the logical form, see 6.g. Examples 7, 13, 18 and Figs. 3, 7, 11, 21, 32. Hence, the present invention provides a set of human anti-HTT antibodies with binding properties ularly useful for diagnostic and therapeutic purposes.
Thus, in one embodiment the present invention provides antibodies which are capable of cally binding pathologically aggregated forms of HTT. However, in addition or alternatively, the antibodies ofthe present invention which are capable to bind to a polyP-region or a P-rich region ofHTT exon 1 may be also utilized in other applications. In particular, these antibodies are not limited to HTT but can also bind to other targets showing also a polyP-tract or a P-rich region.
In one embodiment, the antibody ofthe present invention exhibits the binding properties of the exemplary NI-302.33C11, .63F3, NI-302.35C1, NI.302-7D8 and -72F10 antibodies as described in the Examples. The anti-HTT antibody of the present invention preferentially recognizes pathologically altered HTT, such as mutated and/or aggregated HTT species and fragments thereof rather than physiological HTT. Thus, in one embodiment, the antibody of the present invention does not substantially recognize logical HTT s.
The term "does not ntially recognize" when used in the t application to describe the binding affinity of a molecule of a group comprising an antibody, a fragment thereof or a binding molecule for a specific target molecule, n and/or conformation of the target molecule and/or n means that the molecule of the aforementioned group binds said molecule, antigen and/or conformation with a binding affinity which is at least , 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold less than the binding affinity of the molecule of the aforementioned group for binding another le, antigen and/or conformation. Very often the dissociation nt (KD) is used as a measure of the binding affinity. Sometimes, it is the ECso on a specific assay as for example an ELISA assay that is used as a measure of the binding affinity. Preferably the term "does not substantially recognize" when used in the present application means that the molecule of the aforementioned group binds said molecule, antigen and/or conformation with a binding affinity which is at least or 10—fold, 20-fold, 50-fold, 100- fold, lOOO—fold or lOOOO-fold less than the binding y of said molecule of the aforementioned group for binding to another molecule, antigen and/or conformation.
As described above, the aggregation of HTT in HD is suggested to occur due to an extension of the lutamine tract within the HTT exon 1. In particular, it has been shown that HD mainly occurs in patients having a threshold over the 35-40 glutamine residues in length in the HTT. Accordingly, as shown in Example 3, aggregated and soluble construct of HTT exon 1 with 21, 35 or 49 polyQ repeats were generated in order to identify the utility ofthe anti-HTT- antibodies of the present ion to specifically bind to pathological altered HTT.
The term HDX as used in the following describes the HTT constructs which were ted in accordance with Example 3. Particularly the X denotes the number of glutamine repeats (Qs), e. g. HTT exon 1 protein with 21 polyQ repeats will be denoted HD21.
Utilizing the constructs as described in the Examples, it could be shown that the anti-HTT antibody of the present invention in addition, or alternatively, binds to pathologically, e causing and/or mutated and/or aggregated forms of human HTT. In this context, the binding affinities may be in the range as shown for the exemplary NI-302.33C11, NI-302.63F3, NI- 302.35C1, .31F11, NI-302.6N9, NI-302.46C9, NI-302.8F1, NI-302.74C11, NI- 302.15F9, NI-302.39G12, .11A4, NI-302.22H9, NI-302.44D7, NI-302.55D8, NI- 302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, and NI-302.3D8 dies in Fig. 3(A), 7(A), 11(A), 14(A), tive Fig. 19, 20 and 31, zle. having half maximal effective concentrations (ECso) of about 1 pM to 250 nM, preferably an ECso of about 25 pM to 50 nM, most preferably an ECso of about 0.05 nM to 30 nM for human aggregated HD49-HTT and aggregated recombinant HD49-HTT, or an ECso of about 0.05 nM to 5 nM for human aggregated HD21-HTT and aggregated recombinant HD21-HTT.
In particular, the TT antibody, binding fragment or biotechnological derivative f has a binding affinity corresponding to an ECso value of S 20 nM, preferably 5 10 nM and most preferably 5 1 nM for g aggregated HD49 HTT and/or of S 40 nM, preferably 5 10 nM and most preferably 5 1 nM for binding HD21 HTT; see Fig. 3, 7, 11, 19 and 31.
HTT aggregation associated with the development of HD is most frequently associated with poly—glutamine (polyQ) tracts of > 35 repeats. As shown in the present invention, the anti-HTT antibodies described herein showed high binding efficiency to HD tracts with higher repetitions, see 6.g. Examples 7, 13, 18, 31 and 33. Therefore, in one embodiment of the present invention the anti-HTT antibody, HTT-binding molecule, nt, synthetic or biotechnological variant thereof binds to HTT with expanded poly-glutamine (Q) tract. In a preferred embodiment it binds to HTT with more than 35 repeats. In a particular preferred ment of the present ion, the dy binds to HTT with expanded poly-glutamine (Q) tract consisting of 49 (HD49) repeats over 35 repeats (HD35) and more over 21 repeats (HD21).
However, in accordance with the present invention also anti-HTT antibodies, HTT-binding molecules, fragments, synthetic or biotechnological variants thereof binding to poly-glutamine (polyQ) tracts under 35 (HD35) are described. Therefore, in one embodiment of the present invention, the antibody, g molecule or variants thereof binds to HTT showing l" polyQ tracts. In particular, the antibody is capable of binding to HTT with polyQ tracts < 35 s (HD35).
Some antibodies are able to bind to a wide array of biomolecules, e.g., ns. As the skilled artisan will appreciate, the term specific is used herein to indicate that other biomolecules than HTT ns or fragments f do not significantly bind to the antigen-binding le, e.g., one of the antibodies of the present invention. Preferably, the level of binding to a biomolecule other than HTT results in a binding affinity which is at most only 20% or less, % or less, only 5% or less, only 2% or less or only 1% or less (z'.e. at least 5, 10, 20, 50 or 100 fold lower, or anything beyond that) of the affinity to HTT, respectively; see e.g., Fig. 20.
In one embodiment, the anti-HTT antibody of the present invention binds entially to aggregated forms of HTT and/or nts, derivatives, fibrils and/or oligomers f. In another embodiment the anti-HTT antibody ofthe present invention preferentially binds to both native HTT and pathologically mutated and/or aggregated forms of HTT.
In a further embodiment of the present invention, the anti-HTT antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof is a bispecific antibody. Thus, the antibody of the present invention may be capable of recognizing at least two distinct es either on the same or on different antigens; see also, supra.
In one embodiment, at least one binding site/domain of the bispecific antibody specifically recognizes an e in a polyP-region of HTT, which comprises the amino acid sequence PPPPPPPP (NI-302.33C11; NI-302.44D7; NI-302.7A8; NI—302.3D8; NI-302.46C9) (SEQ ID Nos. 139, 151, 154, 158, 161), amino acid sequence PPPPPP (NI-302.11H6, NI-302.18A1, NI- 302.52C9 (SEQ ID Nos.: 157, 159, 160), amino acid ce PPPPPPPPPPP (NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, .37C12, NI-302.55D8, NI- 302.78H12, NI-302.71F6 (SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156), an epitope in the P-rich-region which ses the amino acid sequence PQPPPQAQPL (NI- 302.63F3 SEQ ID No. 140, NI—302.64E5 SEQ ID No. 200), the amino acid sequence PPPQLPQPPP (NI-302.31F11, SEQ ID No. 141), the amino acid sequence QAQPLLPQPQPPPPP (NI-302.2A2; SEQ ID No. 142), or the amino acid sequence PPPQLPQPPPQAQPL (NI302.15D3; SEQ ID No. 143), an epitope in the C-terminal region which comprise the amino acid sequence PPGPAVAEEPLHRP (NI-302.35C1, SEQ ID No. 145) or PPPGPAVAEEPLH (NI—302.72F10, SEQ ID No. 202), an epitope in the N—terminal region which comprises the amino acid sequence KSFQ (NI-302.15E8, SEQ ID No. 144), an epitope in the ch-region which comprises the amino acid sequence QQQQQQQQQPPP (NI-302.7D8 SEQ ID No. 201), or a conformational epitope recognized by any one of dies N1-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1.
As mentioned before, lation of polyglutamine (poly-Gln, polyQ)-containing HTT protein aggregates in neuronal intranuclear inclusions is a hallmark of the progressive neurodegenerative disorder Huntington's e (HD). Electron raphs of these aggregates revealed fibrillar structures showing a closely related morphology as in B-amyloid fibrils in Alzheimer's disease, see e. g. Caughey et al., Trends Cell Biol. 7 (1997), 56—62 and Caputo et al, Arch. Biochem. Biophys. 292 (1992), 199—205, suggesting that HD, wherein degenerative process ily involves medium spiny striatal neurons and cortical neurons leading to dysfunction and subsequently neuronal loss, tissue damage due to toxicity, mitochondrial damage, free ls, and possibly also inflammatory mechanisms including lia activation and further progressive nature of symptoms, are a result of toxic amyloid fibrillogenesis. Therefore, in one embodiment the antibody of the present invention is useful for the ent of Huntingtion's disease (HD) and ms thereof So far, intracellularly expressed antibodies (intrabodies) have been described and considered as therapeutic tools in HD which perturb the HTT function, see 6.g. Ali et al. in Neurobiology of Huntington's Disease: Applications to Drug Discovery, Lo et al., Chapter 10, CRC Press (2011). Although these intrabodies showed a positive effect on the aggregation and cell death d by HTT in cell based assays, see 6.g. Khoshnan et al., Proc Natl Acad Sci U S A. 99 (2002), 1002—1007, one disadvantage in their therapeutic utility is the route of administration.
In particular, the preferred method for the delivery of the therapeutic intrabodies to the brain is a viral vector-based gene therapy. However, a major disadvantage of using this kind of administration is among other the high host immunogenicity. Therefore, non-viral s utilizing other routes of administration as are preferably used in the therapeutic or diagnostic approaches. The antibodies ofthe present invention have been shown to attenuate the dendritic spine density loss upon addition to the culture medium, z'.e. ellularly. Therefore, in contrast to the intrabodies described before, the antibodies of the present invention can be expected to be efficacious following therapeutically preferred administration routes.
Accordingly, in one embodiment of the t invention the antibody is administrated by a subcutaneous injection (3.0.), intravenous injection (z'.v.), uscular injection (22m), intraperitoneal (2'.p.), intrathecal, jet injection, wherein the radius of action is not limited to the intracellular expression of the antibody.
As already mentioned before, and as shown in Example 24 and Fig. 17 the therapeutic utility ofthe dies of the present invention has been shown. In particular, it has been shown that the anti-HTT antibodies of the present invention are capable of attenuating the dendritic spine density loss. Therefore, in one embodiment of the present invention the ant-HTT dy, the HTT—binding fragment, synthetic or biotechnological tive thereof leads to an attenuation of spine density loss.
Furthermore, the therapeutic utility of the antibodies of the present invention has been demonstrated in Example 34 and Fig. 34. In particular, it has been shown that the TT antibodies of the t invention improve behavioral recovery during task-specific ng and enhance loco-motor ability. ore, in one embodiment of the present invention the ant- HTT antibody, the HTT-binding fragment, synthetic or biotechnological derivative thereof leads to an improvement of behavioral performance during task-specific training and enhancement of sensorimotor ability.
The present invention is also drawn to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody comprises an antigen-binding domain identical to that of an antibody selected from the group consisting of NI-302.33Cll, .63F3, NI- 302.35Cl, NI-302.31Fll, NI-302.2A2, .6N9, .74Cll, NI-302.15F9, NI- 302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37Cl2, NI-302.55D8, NI- 302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18Al, NI-302.8Fl, .52C9, .46C9, NI-302.15E8, NI-302.15D3, NI-302.64E5, NI-302.7D8, NI- 302.72F10, NI-302.12H2, NI-302.8M1 and NI-3024A6.
The present invention further exemplifies several binding les, e.g., antibodies and binding fragments thereof, recognizing a polyP-region of HTT, which may be characterized by comprising in their variable , e.g., binding domain at least one complementarity determining region (CDR) ofthe VH and/or VL variable region comprising any one ofthe amino acid sequences depicted in Fig. l. The ponding nucleotide sequences ng the above- identified variable regions are set forth in Table 11 below. Exemplary sets of CDRs of the above amino acid sequences ofthe VH and/or VL region are depicted in Fig. 1. However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs may be used, which differ in their amino acid sequence from those set forth in Fig. l by one, two, three or even more amino acids in case of CDR2 and CDR3. Therefore, in one embodiment the antibody ofthe present invention or a HTT—binding nt thereof is provided comprising in its variable region at least one complementarity determining region (CDR) as depicted in Fig. 1 and/or one or more CDRs thereof comprising one or more amino acid substitutions.
Further the present invention exemplifies l binding molecules, e. g., antibodies and binding nts f, recognizing the P-rich region of HTT which may be characterized by comprising in their le , e.g., g domain at least one complementarity determining region (CDR) ofthe VH and/or VL variable region sing any one ofthe amino acid sequences depicted in Fig. l. The corresponding tide sequences ng the above- identified variable regions are set forth in Table III below. Exemplary sets ofCDRs ofthe above amino acid sequences ofthe VH and/or VL region are depicted in Fig. 1. However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs may be used, which differ in their amino acid sequence from those set forth in Fig. l by one, two, three or even more amino acids in case of CDR2 and CDR3. Therefore, in one embodiment the antibody ofthe present invention or a HTT-binding fragment thereof is provided sing in its variable region at least one mentarity determining region (CDR) as depicted in Fig. 1 and/or one or more CDRs thereof comprising one or more amino acid substitutions.
The present invention in addition exemplifies several binding molecules, e.g., antibodies and binding fragments thereof, recognizing the C-terminal region of HTT which may be characterized by comprising in their variable region, e.g, binding domain at least one complementarity determining region (CDR) of the VH and/or VL variable region comprising any one ofthe amino acid sequences depicted in Fig. l. The corresponding nucleotide sequences encoding the above-identified variable regions are set forth in Table IV below. Exemplary sets of CDRs of the above amino acid sequences of the VH and/or VL region are depicted in Fig. 1.
However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs may be used, which differ in their amino acid sequence from those set forth in Fig. 1 by one, two, three or even more amino acids in case of CDR2 and CDR3. Therefore, in one embodiment the antibody of the present invention or a HTT-binding nt thereof is provided comprising in its variable region at least one complementarity determining region (CDR) as depicted in Fig. 1 and/or one or more CDRs thereof comprising one or more amino acid substitutions.
Additionally, the present invention exemplifies several binding molecules, e.g., antibodies and binding fragments thereof, recognizing the N—terminal—region of HTT which may be characterized by comprising in their variable , e. g., binding domain at least one complementarity determining region (CDR) of the VH and/or VL variable region comprising any one ofthe amino acid sequences depicted in Fig. l. The ponding tide sequences encoding the above-identified variable regions are set forth in Table VI below. Exemplary sets of CDRs of the above amino acid ces of the VH and/or VL region are depicted in Fig. 1.
However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs may be used, which differ in their amino acid sequence from those set forth in Fig. 1 by one, two, three or even more amino acids in case of CDR2 and CDR3. Therefore, in one embodiment the antibody of the present invention or a HTT-binding nt thereof is provided comprising in its le region at least one complementarity determining region (CDR) as depicted in Fig. 1 and/or one or more CDRs thereof comprising one or more amino acid substitutions.
In one embodiment, the antibody of the t invention is any one of the antibodies comprising an amino acid ce of the VH and/or VL region as depicted in Fig. l or a VH and/or VL region thereof sing one or more amino acid substitutions. Preferably, the antibody ofthe present invention is characterized by the vation of the cognate pairing of the heavy and light chain as was present in the human B-cell.
In a further embodiment of the present invention the anti-HTT antibody, HTT-binding fragment, synthetic or hnological variant thereof can be optimized to have appropriate binding affinity to the target and pharmacokinetic properties. Therefore, at least one amino acid in the CDR or le region, which is prone to modifications selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, iso-aspartate formation and/or unpaired cysteine is substituted by a mutated amino acid that lack such alteration or wherein at least one carbohydrate moiety is deleted or added ally or enzymatically to the antibody. Examples for amino acid optimization can be found in Table VII, wherein antibodies showing primer-induced alterations are shown. Additional modification optimizing the antibody properties are described in Gavel et (11., Protein Engineering 3 (1990), 433-442 and Helenius et al., Annu. Rev. Biochem. 73 (2004), 1019- 1049.
Alternatively, the antibody of the present invention is an dy or antigen-binding fragment, derivative or variant thereof, which competes for binding to HTT with at least one of the dies having the VH and/or VL region as depicted in Fig. 1.
The antibody with at least one dy having the VH and/or VL region as depicted in Fig. 1 competing for binding to HTT may be further characterized in a dot blot assay and/or filter retardation, as bed in Example 6, 13, 18, 31 and/or 32. Therefore, in one embodiment of the present invention the antibody binds to HTT, preferably to HTT with an expanded poly-Q tract consisting of 49 (HD49) s in a dot blot assay and/or filter retardation.
Experimental s provided in Figs. 3, 7, ll, 21, 22 as well as Figs. 32 and 33, and Examples 6, 7, 13, 18, 26 and 32 suggest that some of the anti-HTT antibodies of the present invention preferentially bind to disease causing mutated and/or aggregated forms ofhuman anti-HTT over the other amyloid forming proteins. In one embodiment thus, the antibody of the present invention preferentially recognizes mutated and/or aggregated HTT and/or fragment and/or derivatives f over other amyloid forming proteins.
In one ment of the present invention the TT antibody, HTT—binding fragment, synthetic or biotechnological derivative thereof does preferentially recognize mutated, aggregated and/or soluble forms of HTT over physiological HTT.
The antibody of the present invention may be human, in particular for therapeutic ations.
Alternatively, the dy of the present invention is a rodent, rodentized or chimeric rodent- human antibody, ably a murine, murinized or chimeric murine-human antibody or a rat, ratinized or chimeric rat-human antibody which are ularly useful for diagnostic methods and studies in animals. In one embodiment the antibody of the present invention is a chimeric rodent-human or a rodentized antibody. Furthermore, in one embodiment, the chimeric antibody of the present invention, i.e. comprising the variable domains of a human antibody and generic murine light and heavy nt domains bind with a high affinity to human HTT.
Preferably, the binding affinity of chimeric antibodies is similar to their human counterparts.
In one embodiment the antibody of the present invention is provided by cultures of single or oligoclonal B-cells that are cultured and the supernatant ofthe culture which contains antibodies produced by said B-cells, is screened for presence and affinity of anti-HTT antibodies therein.
The screening process comprises screening for binding to native monomeric, fibrillar or non- fibrillar aggregates like ers of hHTT derived from a tic ength hHTT e or e.g. purified from human plasma or recombinant expression.
As mentioned above, due to its generation upon a human immune response the human monoclonal dy of the present invention will recognize epitopes which are of particular pathological relevance and which might not be accessible or less immunogenic in case of immunization ses for the generation of, for example, mouse monoclonal antibodies and in vitro screening ofphage y libraries, respectively. Accordingly, it is prudent to stipulate that the epitope ofthe human anti-HTT antibody ofthe present invention is unique and no other dy which is capable of binding to the epitope recognized by the human monoclonal antibody of the t invention exists. A further tion for the uniqueness of the antibodies ofthe present invention is the fact that, as indicated in Figs. 19, 20, 24, and 27 to 29, antibodies of the present invention bind epitopes that are specific for the mutated and/or aggregated forms of HTT, which as indicated above, are of particular pathological relevance and may not be obtainable by the usual processes for antibody generation, such as immunization or in vitro library screenings.
Therefore, in one ment the present invention also extends lly to TT antibodies and HTT-binding molecules which compete with the human monoclonal antibody ofthe present invention for specific binding to HTT. The present invention is more specifically directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the dy specifically binds to the same epitope in a polyP-region of HTT as a reference antibody selected from the group consisting of NI-302.33Cl l, NI-302.74Cl l, NI-302.15F9, NI-302.39G12, NI-302.l 1A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI- 302.7A8, NI-302.71F6, NI—302.l 1H6, NI-302.3D8, NI-302.18Al, NI-302.8Fl, NI-302.52C9, NI-302.78H12 and NI-302.46C9. Further, in one embodiment the present invention is more specifically directed to an antibody, or antigen-binding nt, variant or derivatives thereof, where the antibody specifically binds to the same epitope in the -region of HTT as a nce dy selected from the group consisting of NI-302.63F3, NI-302.31Fll, NI- 302.2A2, .15D3 and/or .64E5. In another embodiment the present invention is directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, which binds to the same epitope in the C-terminal—region ofHTT as a reference antibody selected from the group consisting of NI-302.35Cl and/or NI.302-72F10. In a fiirther embodiment the present ion is directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, which binds to the same epitope in the N—terminal-region of HTT as a reference antibody selected from the group consisting of NI-302.15E8. In another embodiment the t invention is directed to an antibody, or antigen—binding fragment, variant or derivatives thereof, which binds to the same epitope of HTT as a reference dy selected from the group consisting ofNI-302.6N9, NI-320.12H2, NI-302.8Ml and/or .4A6. In one embodiment the present invention is directed to an dy, or n-binding fragment, variant or derivatives thereof, which binds to the same epitope ofHTT as reference antibody NI-302.7D8.
In a preferred embodiment the present invention also extends generally to anti-HTT antibodies and nding molecules which e with the human monoclonal dy of the present invention for specific binding to mutated and/or aggregated HTT species or fragments thereof, as shown in Examples 7, 13, 18, 31 and 33 as well as Figs. 7, l3, l9 and 31. The present invention is therefore, more cally also directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the dy specifically binds to the same epitope in a polyP—region ofmutated and/or aggregated HTT species or fragments f as a reference antibody ed from the group consisting of NI-302.74Cll, .15F9, NI- 302.39G12, NI-302.llA4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI- 302.71F6, NI-302.33Cll, NI-302.44D7, NI-302.7A8, NI-302.3D8, .46C9, NI- 302.IlH6, NI-302.18Al, NI-302.52C9, and/or NI-302.8Fl. Further, in one embodiment the present invention is more specifically directed to an antibody, or antigen-binding fragment, variant or derivatives f, where the antibody specifically binds to the same epitope in the P-rich—region of mutated and/or aggregated HTT species or fragments thereof as a reference antibody selected from the group consisting of .63F3, NI-302.3lF1 l, NI-302.2A2, NI- 302.15D3 and/or NI-302.64E5. In another embodiment the present invention is directed to an antibody, or antigen—binding fragment, variant or derivatives thereof, which binds to the same epitope in the C-terminal—region of d and/or aggregated HTT species or fragments thereof as a reference antibody selected from the group consisting of NI-302.35Cl and/or NI.302-72F10. In a further embodiment the present invention is directed to an antibody, or antigen-binding fragment, variant or derivatives thereof, which binds to the same epitope in the N—terminal-region of mutated and/or aggregated HTT species or fragments thereof as a reference dy selected from the group ting ofNI-302.15E8. In another embodiment the present invention is ed to an antibody, or antigen-binding fragment, variant or derivatives thereof, which binds to the same epitope of HTT as a reference antibody selected from the group ting of NI-302.6N9, NI-320.12H2, NI-302.8M1 and/or NI—302.4A6. In one embodiment the present ion is directed to an antibody, or n-binding fragment, variant or biotechnological derivative thereof, which binds to the same epitope of HTT as reference dy NI-302.7D8.
Competition between antibodies is determined by an assay in which the immunoglobulin under test inhibits specific binding of a nce antibody to a common antigen, such as HTT.
Numerous types of competitive binding assays are known, for e: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; see Stahli et (11., Methods in Enzymology 9 (1983), 242-253; solid phase direct biotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phase direct labeled assay, solid phase direct labeled sandwich assay; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); solid phase direct label RIA using I125 label; see Morel er al., Molec. l. 25 , 7-15 and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82.
Typically, such an assay involves the use of purified HTT or mutated and/or aggregated HTT, such as oligomers and/or fibrils thereof bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin, z'. e. the human monoclonal antibody of the present ion. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. ably, the competitive binding assay is med under ions as described for the ELISA assay in the ed Examples. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies g to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50% or 75%. Hence, the present invention is further drawn to an antibody, or antigen-binding fragment, t or derivatives thereof, where the antibody competitively inhibits a reference antibody selected from the group consisting of NI—302.33Cl l, NI-302.63F3, NI-302.35Cl, NI-302.31F11, NI- 302.2A2, NI-302.6N9, NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI- 302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, NI-302.78H12, NI- 302.7lF6, NI-302.l 1H6, NI-302.3D8, NI-302.18Al, NI-302.8Fl, NI-302.52C9, NI-302.46C9, NI-302.15E8, NI-302.64E5, NI-302.7D8, NI-302.72F10, NI-302.12H2, NI-302.8Ml and/or NI-302.4A6 from binding to HTT.
The present invention is further drawn to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody competitively inhibits a reference antibody ed from the group consisting of NI-302.74Cl l, NI-302.15F9, NI-302.39G12, .11A4, NI- 302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6, NI-302.33Cll, NI- 302.44D7, .7A8, NI-302.3D8, NI-302.46C9, NI-302.11H6, NI-302.18Al, NI- 302.52C9, NI-302.8Fl, NI-302.63F3, NI-302.3 lFl l, .2A2, NI302.15D3, NI-302.35Cl, NI-302.6N9, NI-302.7D8 and/or NI-302.72F 10 from binding to mutated and/or aggregated HTT species or fragments thereof.
In a preferred embodiment the antibody, the binding ofan antibody, binding nt, synthetic or biotechnological variant thereof, to HTT, preferably to HTT with an expanded poly-Q tract consisting of 49 (HD49) s can be measured in a dot blot assay and/or filter ation as described in the Examples, in particular in 7, l3, 18, 31 and/or 33.
In another ment, the present invention es an isolated ptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one of VH-CDRs of the heavy chain variable region or at least two of the VH- CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDRZ or VH-CDR3 amino acid sequences from the antibodies disclosed herein. atively, the VH-CDRl, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% cal to reference heavy chain VH -CDR1, VH- CDR2 and VH-CDR3 amino acid sequences from the antibodies sed herein. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDRl, VH-CDR2 and VH—CDR3 polypeptide sequences related to the groups shown in Fig. 1 respectively. While Fig. 1 shows VH-CDRs defined by the Kabat system, other CDR ions, e.g., VH-CDRs defined by the Chothia system, are also included in the present invention, and can be easily identified by a person of ordinary skill in the art using the data presented in Fig. l.
In another embodiment, the present invention provides an isolated polypeptide comprising, ting essentially of, or consisting of an globulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDRZ and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDRZ and VH-CDR3 groups shown in Fig. 1 tively.
In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an globulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDRZ and VH-CDR3 regions have polypeptide sequences which are identical to the l, VH-CDRZ and VH-CDR3 groups shown in Fig. 1 respectively, except for one, two, three, four, five, or six amino acid substitutions in any one VH-CDR. In certain embodiments the amino acid substitutions are conservative.
In another embodiment, the present invention provides an isolated polypeptide sing, consisting ially of, or consisting of an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL- CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDRZ or VL-CDR3 amino acid sequences from antibodies disclosed . Alternatively, the VL-CDRl, VL—CDRZ and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRI, VL'CDR2 and VL- CDR3 amino acid sequences from antibodies disclosed herein. Thus, ing to this embodiment a light chain variable region of the invention has VL—CDRl, VL-CDRZ and VL- CDR3 polypeptide sequences related to the polypeptides shown in Fig. 1 respectively. While Fig. 1 shows VL-CDRs defined by the Kabat system, other CDR definitions, e.g, VL-CDRs defined by the Chothia system, are also included in the present invention.
In another ment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL—CDRZ and VL—CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL'CDR2 and VL-CDR3 groups shown in Fig. 1 respectively. In another ment, the present ion provides an isolated polypeptide comprising, consisting ially of, or consisting of an immunoglobulin light chain le region (VL) in which the VL-CDRl, VL-CDRZ and S regions have polypeptide sequences which are identical to the VL-CDRl, VL'CDR2 and VL-CDR3 groups shown in Fig. 1 respectively, except for one, two, three, four, five, or six amino acid tutions in any one VL-CDR. In certain embodiments the amino acid substitutions are conservative.
An immunoglobulin or its ng cDNA may be further modified. Thus, in a further embodiment the method of the present invention comprises any one ofthe step(s) of producing a chimeric dy, murinized antibody, —chain antibody, Fab-fragment, bi—specific antibody, filSlOIl antibody, d dy or an analog of any one of those. Corresponding methods are known to the person skilled in the art and are described, 6. g. in Harlow and Lane "Antibodies, A Laboratory ", CSH Press, Cold Spring Harbor (1988). When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the ncy of phage antibodies which bind to the same e as that of any one of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; rg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in international ation WO 89/09622. Methods for the production of humanized antibodies are described in, e.g, European application EP-Al 0 239 400 and international application WO 90/07861.
Further sources of dies to be utilized in accordance with the present invention are so- called xenogeneic antibodies. The general principle for the production of xenogeneic antibodies such as human-like antibodies in mice is described in, e. g., ational applications W0 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. As discussed above, the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab)2, as well as in single chains; see 6.g. international application WO 88/09344. In one ment therefore, the antibody of the present invention is provided, which is selected from the group consisting of a single chain FV fragment (scFv), a F(ab') fragment, a F(ab) fragment, and a F(ab')2 fragment.
The antibodies of the present invention or their corresponding immunoglobulin s) can be further modified using conventional ques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY. and Ausubel, Current Protocols in lar Biology, Green Publishing Associates and Wiley Interscience, NY. . Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N— and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the present invention encompasses the production of chimeric proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e.g, ational application WO 00/30680 for corresponding technical details.
The dies of the present invention may also include additional modifications which optimize their therapeutic potential. These modifications comprise but are not limited to modifications to the amino acid sequence of the antibody (e.g, the variable regions) and post- translational modifications. Post-translational modifications (PTMs) are chemical modifications that play a key role in functional proteomics, because they regulate ty, zation and interaction with other cellular molecules such as proteins, nucleic acids, lipids, and cofactors. Therefore, the optimization of the dies may provide several advantages such as an improved stability during storage as well as pharmacokinetics and/or pharmacodynamics profile such as the in vivo or in vitro circulating time of the antibody, increased solubility, stability, sed affinity to the target, decreased te, an improved effector fiinction of the constant region (Fc region) and safety profile ofthe antibody, such as a decreased immunogenicity, or d susceptibility to posttranslational modifications, as shown 6. g. in Igawa et al., MAbs 3 (2011), 243-52. Accordingly, in one embodiment of the present invention the anti-HTT antibody, HTT-binding fragment, synthetic or biotechnological variant f can be optimized, wherein at least one amino acid in the CDR or variable region, which is prone to modifications including but are not limited to ation, acylation, ADP- ribosylation, amidation, deamidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, nt attachment of phosphotidylinositol, cross-linking, cyclization, de bond ion, isomerization, demethylation, formation of covalent cross—links, ion of cysteine, formation of pyroglutamate, formylation, oxylation, glycosylation, GPI anchor formation, hydroxylation, hydrolysis, iodination, methylation, oylation, oxidation, pegylation, proteolytic sing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA ed addition of amino acids to proteins such as arginylation, and ubiquitination (see, e.g., Creighton, "Proteins: Structures and Molecular Properties," 2nd eds, Freeman and Co., NY, 1992; "Postranslational Covalent Modification ofProteins," Johnson, eds., Academic Press, New York, 1983; Seifter et al. Meth.
Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY. Acad. Sei. 663 (1992) 48-62) is substituted by a mutated amino acid that lack such alteration or wherein at least one carbohydrate moiety is d or added chemically or enzymatically to the dy. In a preferred embodiment the modifications are selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, partate formation and/or ed cysteine. Additional modification that optimize the utility of the HTT-antibodies or g molecules as a therapeutic agent are well known in the art and described e.g. in Igawa et al., MAbs 3 (2011), 243-52 which disclosure content is incorporated herein. Means of adding or deleting carbohydrate moieties can be achieved chemically or enzymatically and is described in detail in e. g. Berg et a1. "Biochemistry" 5th eds W H Freeman, New York 2002; WO 87/05330; Aplin et al., CRC Crit. Rev. Biochem., 22 (1981), 259-306; Hakimuddin et al., Arch.
Biochem. s, 259 (1987), 10-52; Edge et al., Anal. Biochem., 118 (1981), 131; Thotakura et al., Meth. Enzymol. 138. (1987), 350. onally, the present invention encompasses peptides including those containing a binding molecule as described above, for example containing the CDR3 region of the variable region of any one of the ned antibodies, in ular CDR3 of the heavy chain since it has frequently been observed that heavy chain CDR3 (HCDR3) is the region having a greater degree of variability and a predominant participation in antigen—antibody interaction. Such peptides may easily be synthesized or produced by recombinant means to produce a binding agent useful according to the invention. Such methods are well known to those of ordinary skill in the art. Peptides can be synthesized for example, using automated peptide synthesizers which are commercially available. The peptides can also be produced by recombinant techniques by incorporating the DNA expressing the peptide into an expression vector and orming cells with the expression vector to produce the peptide.
Hence, the present invention relates to any binding molecule, e.g., an antibody or binding fragment thereof which is oriented towards the TT antibodies and/or antibodies e of binding mutated and/or aggregated HTT species and/or fragments thereof of the present invention and displays the mentioned properties, 2'. e. which specifically recognizes HTT and/or mutated and/or aggregated HTT species and/or fragments f Such antibodies and g molecules can be tested for their binding specificity and affinity by ELISA and immunohistochemistry as described herein, see, e.g., the Examples. These characteristics of the antibodies and binding molecules can be tested by Western Blot as well.
As an alternative to obtaining immunoglobulins directly from the e of B cells or memory B cells, the cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation. nged antibody genes can be reverse ribed from appropriate mRNAs to e cDNA. If desired, the heavy chain nt region can be exchanged for that of a different isotype or eliminated altogether. The variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed. Once the genetic material is available, design of analogs as described above which retain both their ability to bind the desired target is straightforward. Methods for the cloning of antibody variable regions and generation of recombinant antibodies are known to the person skilled in the art and are described, for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke er al., Leukemia 11 (1997), 1787-1792.
Once the riate genetic material is obtained and, if desired, modified to encode an analog, the coding sequences, ing those that encode, at a minimum, the variable regions of the heavy and light chain, can be inserted into expression systems contained on vectors which can be transfected into standard inant host cells. A variety of such host cells may be used; for efficient processing, however, mammalian cells are preferred. l mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.
The production of the antibody or analog is then undertaken by culturing the modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences. The antibodies are then recovered by isolating them from the culture. The sion systems are ably designed to e signal es so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.
In accordance with the above, the present invention also relates to a polynucleotide encoding the antibody or equivalent binding molecule of the present invention, in case of the antibody preferably at least a variable region of an immunoglobulin chain of the antibody described above. Typically, said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable region of the said antibody.
The person skilled in the art will readily appreciate that the variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. Thus, the present ion also encompasses polypeptides and dies comprising at least one CDR of the above—described variable domain and which advantageously have substantially the same or similar g properties as the antibody described in the appended examples. The person skilled in the art knows that binding affinity may be enhanced by making amino acid substitutions within the CDRs or within the hypervariable loops (Chothia and Lesk, J. Mol.
Biol. 196 (1987), 901-917) which partially overlap with the CDRs as defined by Kabat; see, e.g., Riechmann, er al, Nature 332 (1988), 323-327. Thus, the present invention also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more, preferably not more than two amino acid tutions. Preferably, the antibody ofthe invention ses in one or both of its globulin chains two or all three CDRs of the variable regions as set forth in Fig. 1.
Binding molecules, 6.g. , dies, or antigen-binding fragments, tic or biotechnological variants, or derivatives thereof of the ion, as known by those of ordinary skill in the art, can comprise a constant region which mediates one or more effector filnctions. For example, binding of the C1 ent of complement to an antibody constant region may activate the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory se and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to receptors on s cells Via the Fc region, with a Fc receptor binding site on the dy Fc region binding to a PC receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma ors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, nce of immune complexes, lysis of antibody- coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
Accordingly, certain embodiments of the present invention include an antibody, or antigen- binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been d or otherwise altered so as to provide desired mical characteristics such as reduced effector fianctions, the ability to non-covalently dimerize, increased ability to localize at the site of HTT aggregation and deposition, reduced serum half-life, or increased serum ife when compared with a whole, unaltered antibody of approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an globulin heavy chain, but which lack at least a portion of one or more heavy chain domains. For instance, in certain antibodies, one entire domain ofthe constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted. In other embodiments, certain antibodies for use in the diagnostic and ent methods described herein have a constant region, e.g, an IgG heavy chain constant region, which is altered to eliminate glycosylation, referred to elsewhere herein as aglycosylated or "agly" antibodies. Such "agly" antibodies may be ed enzymatically as well as by engineering the consensus glycosylation site(s) in the constant region. While not being bound by theory, it is believed that "agly" antibodies may have an improved safety and stability profile in viva. s ofproducing aglycosylated antibodies, having desired or fiinction are found for example in international application , which is incorporated by reference in its entirety.
In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated to decrease effector on using techniques known in the art. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody y sing HTT localization. In other cases it may be that nt region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated xin. Yet other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to sed antigen specificity or antibody lity. The resulting physiological , bioavailability and other biochemical effects of the modifications, such as HTT localization, biodistribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be d or exchanged for alternative protein sequences to increase the cellular uptake of antibodies by way of example by ing or-mediated endocytosis of antibodies Via Fcy receptors, LRP, or Thyl receptors or by 'SuperAntibody Technology', which is said to enable dies to be shuttled into living cells without harming them (Expert Opin. Biol. Ther. (2005), 23 7-241). For example, the generation of filSlOIl proteins of the antibody binding region and the cognate protein ligands of cell e receptors or bi- or multi-specific dies with a specific sequences binding to HTT as well as a cell surface receptor may be engineered using techniques known in the art.
In certain antibodies, or antigen-binding nts, variants, or derivatives thereof described herein, the Fc portion may be mutated or exchanged for alternative protein sequences or the antibody may be ally modified to increase its blood brain barrier penetration.
Modified forms of antibodies, or n—binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. Antibodies, or n- binding fragments, variants, or derivatives thereof of the invention can be made or manufactured using techniques that are known in the art. In certain embodiments, antibody molecules or nts thereof are "recombinantly produced", z'.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
Antibodies, or antigen-binding fragments, variants, or tives thereofof the invention also include tives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not by way of tion, the antibody derivatives include antibodies that have been modified, e. g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, tization by known protecting/blocking groups, proteolytic ge, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific al cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. onally, the derivative may contain one or more non-classical amino acids.
In particular preferred embodiments, dies, or antigen-binding fragments, variants, or derivatives thereof of the invention will not elicit a deleterious immune se in the animal to be d, e.g., in a human. In certain embodiments, binding molecules, e. g., antibodies, or n-binding fragments thereof of the invention are derived from a patient, e.g, a human patient, and are subsequently used in the same species from which they are derived, e.g. , human, alleviating or minimizing the occurrence of deleterious immune responses.
De-immunization can also be used to decrease the imrnunogenicity of an antibody. As used , the term "de—immunization" includes alteration of an antibody to modify T cell epitopes; see, e.g., international applications WO 76 and WO 00/34317. For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity determining s (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid tutions and these sequences are uently incorporated into a range of binding polypeptides, e.g., HTT—speciflc antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production ofwhole dy. The antibodies are then compared in appropriate biochemical and biological , and the optimal variant is identified.
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal dies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al., in: Monoclonal dies and T-Cell Hybridomas Elsevier, N.Y., 563-681 (1981), said references incorporated by reference in their entireties. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, yotic, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. In certain embodiments, dies of the present invention are derived from human B cells which have been immortalized Via ormation with Epstein-Barr Virus, as described herein.
In the well-known hybridoma process (Kohler et al., Nature 256 (1975), 495) the relatively short-lived, or mortal, lymphocytes from a mammal, e.g. , B cells derived from a human subject as described herein, are filSCd with an immortal tumor cell line (e.g. a a cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody ofthe B cell. The ing hybrids are segregated into single genetic strains by selection, dilution, and re-growth with each individual strain comprising specific genes for the formation of a single antibody. They e antibodies, which are homogeneous against a desired antigen and, in nce to their pure genetic parentage, are termed "monoclonal".
Hybridoma cells thus ed are seeded and grown in a suitable culture medium that preferably ns one or more substances that inhibit the growth or al of the unfused, parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially ble from a number ofsources and standardized protocols are well established. Generally, culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen. The binding city of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) as described herein. After hybridoma cells are identified that produce antibodies of the desired city, affinity and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods; see, e.g., Goding, Monoclonal dies: Principles and Practice, Academic Press (1986), 59-103. It will further be appreciated that the monoclonal antibodies secreted by the subclones may be separated from culture medium, s fluid or serum by tional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
In another embodiment, lymphocytes can be selected by micromanipulation and the variable genes ed. For example, peripheral blood clear cells can be ed from an immunized or naturally immune mammal, e.g. a human, and cultured for about 7 days in vitro.
The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig—producing B cells can be ed by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be anipulated into a tube and the VH and VL genes can be amplified using, e. g., RT- PCR. The VH and VL genes can be cloned into an dy expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression. atively, antibody-producing cell lines may be selected and cultured using techniques well known to the skilled n. Such techniques are described in a y of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
Antibody fragments that recognize specific epitopes may be generated by known techniques.
For example, Fab and F(ab')2 fragments may be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab nts) or pepsin (to e F(ab')2 fragments). F(ab')2 fragments contain the variable , the light chain constant region and the CH1 domain ofthe heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portions of globulins to detecting reagents such as radioisotopes.
In one embodiment, an antibody of the ion comprises at least one CDR of an antibody molecule. In another embodiment, an dy of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antibody of the ion comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antibodies are described herein.
Antibodies of the present invention can be produced by any method known in the art for the sis of antibodies, in particular, by chemical synthesis or preferably by recombinant expression techniques as described herein.
In one embodiment, an antibody, or antigen—binding nt, variant, or derivative thereof of the invention comprises a synthetic constant region wherein one or more domains are partially or ly deleted ("domain—deleted dies"). In certain ments compatible modified antibodies will comprise domain deleted constructs or ts wherein the entire CH2 domain has been removed (ACH2 constructs). For other embodiments a short ting peptide may be substituted for the deleted domain to provide flexibility and m of movement for the variable region. Those skilled in the art will appreciate that such constructs are particularly preferred due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody. Domain d constructs can be derived using a vector encoding an IgG1 human nt domain, see, e. g., ational applications WO 02/060955 and WO 02/096948A2.
This vector is engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgG1 constant region.
In certain embodiments, dies, or antigen-binding fragments, variants, or derivatives thereof of the t invention are minibodies. Minibodies can be made using methods described in the art, see, e.g., US patent 5,837,821 or international application WO 94/09817.
In one embodiment, an dy, or antigen-binding fragment, variant, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomeric subunits. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase HTT localization.
Similarly, it may be desirable to simply delete that part of one or more constant region domains that control the effector function (e. g. complement binding) to be modulated. Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant s of the disclosed antibodies may be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (6. g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Yet other embodiments comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as an effector fimction or provide for more cytotoxin or carbohydrate attachment. In such embodiments it may be desirable to insert or replicate specific ces d from selected nt region domains.
The present invention also provides antibodies that se, consist essentially of, or consist of, variants (including derivatives) of dy molecules (e.g, the VH s and/or VL regions) described herein, which antibodies or fragments thereof immunospecifically bind to HTT. Standard ques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody, ing, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions.
Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid tutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid tutions, or less than 2 amino acid substitutions relative to the reference VH region, VH-CDRl, VH-CDR2, VH-CDR3, VL region, VL-CDRl, VL-CDRZ, or VL-CDR3. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, phan), beta- ed side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain ty (e. g., the y to bind HTT and/or mutated and/or aggregated HTT species and/or fragments thereof).
For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations may be silent or neutral missense mutations, e.g., have no, or little, effect on an antibody's ability to bind antigen, indeed some such mutations do not alter the amino acid sequence whatsoever. These types ofmutations may be usefiJl to optimize codon usage, or improve a hybridoma's antibody tion. Codon— optimized coding regions encoding antibodies of the t invention are disclosed elsewhere herein. Alternatively, non-neutral missense mutations may alter an antibody’s ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral se ons is likely to be in CDR, though this is not an absolute requirement. One of skill in the art would be able to design and test mutant molecules with desired ties such as no tion in n-binding activity or alteration in g activity (e.g., improvements in antigen-binding activity or change in antibody specificity). Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospeciflcally bind at least one epitope of HTT and/or mutated and/or aggregated HTT species and/or fragments thereof) can be determined using techniques described herein or by routinely modifying techniques known in the art. 111. Polynucleotides ng Antibodies A polynucleotide encoding an antibody, or n-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be fied RNA or DNA or modified RNA or DNA. For example, a cleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be -stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions.
In addition, a polynucleotide encoding an dy, or n-binding fragment, variant, or derivative thereof can be composed of triple—stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an dy, or antigen-binding fragment, variant, or derivative thereofmay also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be d by ucing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded n. Mutations may be introduced by rd techniques, such as site-directed mutagenesis and diated mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
As is well known, RNA may be isolated from the original B cells, hybridoma cells or from other transformed cells by standard techniques, such as a inium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art. In one embodiment, cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well-known methods. PCR may be initiated by consensus constant region primers or by more c primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones ng the antibody light and heavy chains. In this case the libraries may be screened by consensus s or larger homologous probes, such as human constant region probes. DNA, lly plasmid DNA, may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with rd, well known techniques set forth in detail, e.g., in the ing references relating to recombinant DNA techniques. Of , the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
In this context, the present invention also relates to a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody of the present invention. In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain le region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90%, or 95% cal to reference heavy chain VH-CDRI, VH-CDRZ, or VH- CDR3 amino acid sequences from the antibodies disclosed herein. Alternatively, the VH-CDRI, VH-CDRZ, or VH-CDR3 regions of the VH are at least 80%, 85%, 90%, or 95% identical to reference heavy chain VH-CDRI, VH-CDRZ, and VH-CDR3 amino acid sequences from the antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDRI, VH-CDRZ, or VH-CDR3 polypeptide ces d to the polypeptide sequences shown in Fig. 1.
In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRS of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90%, or 95% identical to reference light chain VL-CDRI, VL-CDRZ, or 3 amino acid sequences from the antibodies disclosed herein. Alternatively, the VL-CDRl, VL-CDRZ, or 3 regions ofthe VL are at least 80%, 85%, 90%, or 95% identical to reference light chain VL-CDRl, VL- CDR2, and VL-CDR3 amino acid sequences from the antibodies disclosed herein. Thus, according to this embodiment a light chain variable region of the invention has VL-CDRl, VL- CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in Fig. 1.
In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDRI, VH-CDRZ, and VH-CDR3 regions have ptide ces which are identical to the VH-CDRI, VH-CDRZ, and VH-CDR3 groups shown in Fig. 1.
As known in the art, "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide. When discussed herein, whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to r polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program nsin Sequence is Package, Version 8 for Unix, Genetics Computer Group, sity Research Park, 575 Science Drive, Madison, WI .
T uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the best segment ofhomology between two sequences.
When using BESTFIT or any other sequence alignment program to determine whether a ular sequence is, for example, 95% identical to a reference sequence according to the t invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology ofup to 5% of the total number of amino acids in the reference sequence are allowed.
In a preferred embodiment of the present ion, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide ce of the VH or VL region of an anti-HTT antibody and/or dy recognizing a polyP-region in the HTT and/or mutated and/or aggregated HTT species and/or fragments thereof as depicted in and Table 11.
Additionally, in one embodiment the polynucleotide comprises, ts essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti- HTT antibody and/or antibody recognizing the P-rich—region in the HTT and/or mutated and/or aggregated HTT s and/or fragments thereof as depicted in and Table III and/or further recognizing the C-terminal region in the HTT and/or mutated and/or aggregated HTT species and/or fragments thereof as depicted in and Table IV and/or further recognizing the Q/P-rich region of HTT and/or mutated and/or aggregated HTT species and/or fragments thereof as depicted in Table VII. In addition, in one embodiment the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti-HTT antibody and/or dy recognizing the HTT and/or mutated and/or aggregated HTT species and/or nts thereof as depicted in and Table V. Furthermore, in one embodiment the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti-HTT antibody and/or antibody recognizing the inal region in the HTT and/or d and/or aggregated HTT species and/or fragments thereof as depicted in and Table VI. In addition, in one ment the polynucleotide comprises, ts essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti-HTT antibody and/or antibody recognizing the HTT and/or mutated and/or aggregated HTT species and/or fragments thereof as depicted in and Table V. Additionally or alternatively, in one embodiment the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti-HTT antibody and/or antibody as depicted in and Table VIII.
In this respect, the person skilled in the art will readily appreciate that the polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domain of both globulin chains or only one. In one embodiment therefore, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH and the VL region of an anti-HTT antibody and/or fragments thereof as depicted in Table II, III, IV, V, VI, VII or VIII.
Table II: Nucleotide sequences of the VH and VL region of antibodies recognizing an epitope of a polyP-region of HTT, z'.e exon 1 in aggregated form.
Antibody Nucleotide ces ofvariable heavy (VH) and variable light (VL) chains NI-302.33C11- VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGGGAACTCC CTGAGACTCTCCTGTGCAGCGTCTGGATTCAGGTTCAGTGACTTTGGCATGC ACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGCTGGCACTTATAT GGTATGATGGAGGGTATAAGTACTATGCAGACTCCGTGAAGGGCCGATTCA CCATCTCCAGAGACAATTCCAAGAATACGATGTTTCTACAAATGAACAGCCT GAGAGCCGAGGACACGGCTGTTTATTACTGTGCGACCCACCTAGAATATTGC AGTAGAACCACCTGCTATCTCGGCCACTGGGGCCAGGGAACCCTGGTCACC SEQ ID NO: 1 NI-302.33C11- VK GACATCCAGTTGACCCAGTCTCCGTCCTTCCTATCTGCGTCTGTGGGAGACA CCTTCACTTGCCGGGCCAGTCAGGGCATTAGCGATTATTTAGCCTG GTTTCAGCAGAAACCAGGGATTGCCCCTAAGCTCCTGATCTATGCTGCGTCC ACTTTGCAAACCGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACA GAATTCACTCTCACAATCCGCAGCCTGCAGTCTGAAGATTTTGGAACTTATT ACTGTCAGCAGCTTAAAACTTACCCGTACACTTTTGGCCAGGGGACCAAGGT GGAAATCAAA SEQ ID NO: 3 NI-302.74C11- VH GAGGTGCAGCTGGTGCAGTCTGGGACTGAGGTGCAGAAGCCTGGGGCCTCA GTAAAAGTCTCCTGCAAGGCTTCTGGATACAGTTTCACCGGCTACTTTTTGC ACTGGGTACGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGTGGATCA ACCCTAACAGTGGTGACACAAACTATGCAGAGAAGTTTCGGGGCAGAATCA TCATGACCAGGGACACGTCTGTCAGCACAGCCCACATGGAGTTGAGCAGCC TGAGATTTGACGACACGGCCCTATATTACTGTACGAGAGAGGCCCCTGACCC GGGCGCTGAGACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC SEQ ID NO: 25 NI—302.74C1 1- VL CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGG CCAGGATCACCTGCTCTGGAGATGCAGTGCCAAAGCAGTATATTTATTGGTA CCAGCAGAAGCCAGGCCAGGCCCCTATTCTGGTGATATATAAAGACACTCA GAGGCCTTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCAGGGACAACA GTCACGTTGACCATAACTGGCGTCCAGGCAGACGACGAGGGTGACTATTAC TGTCAATCAGCAGACAGTAGTGCTACTTGGGTGTTCGGCGGAGGGACCAAA TTGACCGTCCTA SEQ ID NO: 27 NI-302.15F9- VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCACGCCGGGGGGGTCC CTGAGACTCTCGTGTGAGGCCTCTGGATTTCTCTTCAAGAATTCTAGCATGA TCCGTCAGACTCCGGGGAAGGGGCTGGAGTGGGTCTCGTCCATTG CTGCTACAAATTATAAGTATTATGCAGACTCTGTGAAGGGCCGATT TACCATCTCCAGGGATGACGCCACCAACTCTCTCTATCTGCAAATGAATAGC CTGCGAGCCGACGACACGGCTACTTATTACTGTGCGCGAGGTTATTATACCC CCCGGGACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 29 NI-302.15F9— VK GATGTTGTGATGACTCAGTCTCCACAGACCCTGTCCGTCAGCCTTGGACAGG CGGCCTCCATCTCCTGCAGGTCGAGTCAAAGCCTCTTGTATCGTGATAACAA CACATACTTGAATTGGTTTCACCAGAGGCCAGGCCAATCTCCAAGGCGCCTC ATTTATAGGGCTTCTGACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCG GTGGGTCAGGCACTGATTTCACATTGAAAATCAGTGGAGTGGAGGCTGAAG ATGTTGGCACTTATTACTGCATGCAAGGAACACACTGGCCTCGGACGTTCGG CCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 31 NI-302.39G12- VH CAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCACCCTTGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTAATTACGCCATAA CTTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGCAATATATTTCAGTAATTTA TCGTGATGGCAGGACATACTACGGAGACTCCGTGAGGGGCCGCTTCACCAT CTCTAGGGACGATTCCAAGAACACTCTCTATCTTCAAATGAACAGCCTGAGA TTTGAGGACACGGCTGTGTATTACTGTGCGAGAGCGCACGGCCAATATTACT ATGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 33 .39G12- VK GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCAGCCCTGGAGAGC CGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGATA CAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAGCTCCTG ATCTATTTGAGTTCTAATCGGCCCTCCGGGGTCCCTGATAGGTTCAGTGCCA GTGGATCAGGCACAGAGTTCACACTGCAAATCAGCAGAGTGGAGGCTGAGG ATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGG AGGGACCAAAGTGGATATCAAA SEQ ID NO: 35 NI-302.11A4- VH GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCGGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGGTTCCCCGTCAGTAGCAGTTACATGA GCTGGGTCCGCCAGGCTCCAGGAGAGGGGCTGGAGTGGGTCTCAGTTCTTTA TAGAGACGGTGACACATACTACGCAGACTCCGTGCAGGGCCGATTCACCAT CTCCAGAGACAATTCCCAGAACACGTTCTATCTTCAAATGAACAGCCTGAAA GACACGGCCGTGTATTACTGTGCGGGTGATAGAAGGTCGTCACAC TACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCT SEQ ID NO: 37 NI-302.11A4- VK GAAATTGTGATGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGAGAAA GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTCGC CTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTACG TCCCGCAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGG ACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGT ATTACTGTCAACAGTATGGTAGCTCGTGGACGTTCGGCCCAGGGACCAAGGT GGAGATCAAA SEQ ID NO: 39 NI-302.22H9— VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCACCCTTGGGGGTCC CTGAGAGTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTAATTACGCCATAA CTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATATATTTCAGTGATTTA TCGTGATGGCAGGACATACTACGGAGACTCCGTGAGGGGCCGCTTCACCAT CTCTAGGGACGATTCCAAGAACACTATCTATCTTCAAATGAACAGCCTGAGA TTTGAGGACACGGCTGTGTATTACTGTGCGAGAGCGCACGGCCAATATTATT ATGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO 41: NI-302.22H9- VK GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCAGCCCTGGAGAGC CGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGATA CAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAACTCCTG ATCTATTTGAATTCTAATCGGGCCTCCGGGGTCCCTGATAGGTTCAGTGGCA GTGGATCAGGCACAGAGTTCACACTGACAATCAGCAGAGTGGAGGCTGAGG ATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGG AGGGACCAAGGTGGAGATCAAA SEQ ID NO: 43 NI-302.44D7- VH GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGA GTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTG GTTATAGTGATACTAGCACATATTACGCAGACTCCGTGAAGGGCCGCTTCAC CGTCTCCAGAGACATTTCCAAGAACACGCTGTATCTGCAAATGAATAGCCTG AGGGCCGAGGACACGGCCGTATATTACTGCGCGAAAGGTACCAGGGACTAT TACGGTATGGACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO: 45 NI-302.44D7- VK CAGACTGTGGTGACTCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAG TCACACTCACTTGTGGCTTGAGTTCTGGCTCAGTTTCTACTAGTTACTACCCC AGCTGGTACCAGCAGACCCCAGGCCGGGCTCCACGCACGCTCATCTACAGC ACAAACACTCGCTCTTCTGGGGTCCCTGATCGCTTCTCTGGCTCCATCCTTGG GAACAAGGCTGCCCTCACCATCACGGGGGCCCAGGCAGATGATGAATCTGA CTGTGTGCTGTTTATGGGTAGTGGCATTGGGGTGTTCGGCGGAGGG ACCAGGCTGACCGTCCTA SEQ ID NO: 47 NI—302.37C12- VH GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTGGTCCAGCCTGGGGGGTCC CTGAGACTCTCTTGTGTTGCCTCTGCACTCACCGTCACTAACAGCCAAATGA CCTGGGTCCGCCGGGCTCCAGGGAGGGGGTTGGAGTGGGTCTCAGTTATTTA CACCAGTGGTAGTGCATACTACGCAGACTCCGTGAAGGGCAGATTCACCAT CTCCAGAGACAATTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAG AGTCGAAGACACGGCTGTGTATTACTGTGCGAAAGGCCCATCAGCCTATTAT TACGGTTTGGACCTTTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 49 NI-302.37C12- VK GATATTGTGATGACTCAATCACCACTCTCCCTGCCCGTCACCCCTGGAGAGC CGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCGGGGCAGTCTCCACAGCTCCTG ATCTATTTGGGTTCTACTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCACAGATTTTACACTGAAGATCAGCAGAGTGGAGGCTGAGG ATGTTGGGGTTTATTACTGCATGCAAGGTCTACAGACGTACACTTTTGGCCA GGGGACCAAGCTGGAGATCAAA SEQ ID NO: 51 NI—302.55D8- VH CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAAGAAGCCTGGGGCCTCA GTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGACTACTATATAC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGACGGATCA ACCCTAACAATGGTGGCACAAACTATGCACAGAACTTTCAGGGCTGGGTCA CCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTCAGCAGAC TGAGATCTGACGACACGGCCGTCTATTACTGTGCGAGAGTGGGGGGCGAGC TGCTACGAGAAGGCGGCTATCACTACTACATGGACGTCTGGGGCAAGGGGA CCACGGTCACCGTCTCCTCG SEQ ID NO: 53 NI-302.55D8- VL CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGG ATCTCCTGCACTGGGAACAGCTCCAACATCGGGGCAGGTTATGATG TACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTTTGA TAATACCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT GGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTA ATTATCACTGCCAGTCCTATGACAACAGCCTGAGTGGTTCTTGGGTGTTCGG CGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 55 .7A8- VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTCCAGCCTGGGGGGTCC CTGAGACTCTCCTGTGTAGCCTCTGGATTCATATTTAGAAACAGTTGGATGA CCTGGGTCCGCCAGGATCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAA AGGAAGATGGAAGTCGGACATACTATGTGGACTCTGTGAAGGGCCGATTCA CCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAGATGAACAGCC TGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGGAGATTATAATT CGGGCATCTATTACTTTCCCGGGGACTACTGGGGCCAGGGCACCCTGGTCAC CGTCTCCTCG SEQ ID NO: 57 NI—302.7A8- VK GTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGC CGGCCTCCATCTCCTGTAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAA CACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAGTCTCCAAGGCGCCTC ATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCA CAGGCACTGATTTCACACTGAGAATCAGCAGGGTGGAGGCTGAGG ATGTTGGCATTTATTACTGCATGCAAGGTACACACTGGCCTGGGACGTTCGG CCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 59 NI-302.78H12- VH CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACC CTGTCCCTCACCTGTCTTGTCTCTAGTTACTCCATCAGCAATGGTTACTACTG GGGCTGGATTCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTAT CTATCATAATGGGAACACCTATTACAACCCGTCCCTCAAGAGTCGAGTCATC ATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGTTGAGGTCTGTGA CCGCCGCAGACACGGCCGTGTACTACTGTGCGATGCCAAGTGCCACCTATTA TTATGGTTCGGGGACTCAATTCCATGCGTTTGATGTCTGGGGCCAAGGGACC ACGGTCACCGTCTCCTCG SEQ ID NO: 61 NI-302.78H12- VL CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAG TCACCATCTCCTGCACTGGAACCAGCAGAGATGTTGGTAATTATAACTATGT CTCCTGGTACCAACAACACCCAGGCGAAGTCCCCAAACTCATAATTTATGAT GTCAGTGAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTG GCAACACGGCCTCGCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTG ACTATTACTGCTGCTCATATGCTGGCAGTTACACCTTCGAGGTATTTGGCGG AGGGACCAAGCTGACCGTCCTA SEQ ID NO: 63 NI-302.71F6- VH CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTATTGAAGCCTTCGGAGACC CTGTCCCTCACGTGCGCTGTCTATGGTGGGTCCCTCAGTGGTTACTACTGGA GCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATAGGGGAAGTCA ATCATAGTGGAGGCACCAACCTCAATTCGTCCCTCAAGAGTCGAGTCATCAT AGACAAGTCCAAGAAGCAGTTCTCCCTGAAACTGAGCTCTGTGACC GCCGCGGACACGGCTATGTACTTCTGTGCGAGAGGATACAGCTATGACCCA AAATACTACTTTGACTCCTGGAGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 65 NI-302.71F6- VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGGCGA TCACCATCTCCTGCACTGGAACCAGTAGTGATATTGGGAGTTATGATTTTGT GTACCAGCAGGACCCAGGCAAAGCCCCCAAAGTCATTATTTATGGG GTCAATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTG GCAACACGGCCTCCCTGACAATCTCTGGACTCCAGGCTGACGACGAGGCTG ATTATTACTGCTGCTCATATGCTGGTAGTACCACTTGGGTGTTCGGCGGAGG GACCAAACTGACCGTCCTA SEQ ID NO: 67 NI-302.11H6- VH GAGGTGCAGCTGGTGCAGTCTGGAGCTGTGATGAAGAAGCCTGGAGACTCA GTGAGGGTCTCCTGCAGGGCTTCTACTTACAGCTTTTCCACCTATAGTTTCAC CTGGGTGCGACAGGTCCCTGGACAAGGCCTTGAGTGGATGGGATGGATCAG CGCTTATAATGGTCACACAAACTATGTAGACAGCTTCCAGGGCAGACTCACG TTGACCACAGACACATCCGCGAGTACAGCGTACATGGAACTGAGGAGCCTC AGATCTGACGACACGGCCATCTATTATTGTGCGGCTGTAGACACCACTTACT ACTATTACGGCATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTC SEQ ID NO: 69 NI-302.11H6— VL CAGACTGTGGTGACTCAGGAGCCAACGTTCTCAGTGTCCCCTGGAGGGACA GTCACACTCACTTGTGCCTTGAGGTTTGGCTCAGTCTCTAGTAGCTACTATCC GTTCCAGCAGACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGC ACAAACACCCGCTCTTCGGGGGTCCCTGCTCGATTCTCTGGCTCCATTCTTGG AGCTGCCCTCACCATCGCGGGGGCCCAGGCAAATGATGAGGCTGA CTATTACTGTGTGCTGTATATGGGTAGTGGAATCGGGGTGTTCGGCGGAGGG ACCAAGTTGACCGTCCTA SEQ ID NO: 71 NI-302.3D8- VH GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC CTGAGACTCTCTTGTGAAGCCTCCGGATTCATCTTTAAAACCTATGCCATGA GCTGGGTCCGCCAGCTTCCCGGGAGGGGGCTGGAATGGGTCTCAGCTATAA GTGCCACTGGTGGAAGCACCTTCTACGCAGAGTCCGTGAAGGGCCGGCTCA CCATTTCCAGAGACACTGCCAAGAATACAGTGTATCTGCAAATGAACAACCT GAGAGCCGAAGACACGGCCATGTATTACTGTGCGAAAGGGTCGACTGCGGT ATATCTCTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 73 .3D8— VK GACATCCAGATGACCCAGTCTCCGTCCTCACTGTCTGCATCTGTAGGGGACA GAGTCACCCTCACTTGTCGGGCGAGTCAGGACATCAGAAATTTCTTGGCCTG GATTCAGCAGAAGCCAGGGAAACCCCCTAAGTCCCTGATCTATGCTGCGTCC ACTTTGCAAAGTGGGGTCCCATCACGATTCAGCGGCAGTGGATCCGGGACA GATTTCACTCTCACCATCAGCAGCCTGCACCCTGAAGATTTTGCTACTTATTA CTGCCAGCAGTTTTATAATTACCCTCCGACGTTCGGCCAAGGGACCAAGGTG GAGATCAAA SEQ ID NO: 75 NI-302.18A1- VH CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTAGTGAAGCCTTCGGAGGCC CTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCACTACTGATTATTACTA TTGGGGCTGGATCCGCCAGTCCCCAGGCAAGGGACTAGAGTGGGTTGGGAC AATATACTTTGGTGGGGCCACCTACTACAATCCGTCCCTCAGGAACCGGGTC TCGATATCTGTGGACACGTCCAACACTCGCCTCTCCCTGAGACTTATCTCTCT GAGCGCCGCTGACACGGCCGTCTATTATTGTGCGAGAGTGGGCTACTTGGAT AGGAGTGGTCTTCTTGTGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 77 .18A1- VK GAAATTGTGCTGACGCAGTCTCCACTCTCCGTGCCCGTCACCCCCGGAGAGC CGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAATAATGGATA CAACTATTTGGATTGGTACCTGAAGAAGCCTGGGCAGTCTCCACAACTCCTG ATCTATTTGGGCTCTACTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGCCA GTGGATCAGGCACAGACTTTACACTGGAAATCAGCAGAGTGGAGGCTGAAG ATGTTGGCGTTTACTACTGCATGCAAGCTCTGCAGACTCCTCCGACTTTCGG CAGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 79 NI-302.52C9- VH GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAACCTGGGGGGTCC CTGAGACTCTCCTGTGCAGGCTCTGGATTCACCGTCAGTGACACCTACATGA GTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTC ATGCCGGTGGTGAAACATATTACGCAGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACTCCAAGAACACGCTGTATCTTCAAATGAATAGGCTGAC GGACACGGCTGTCTTTTATTGTGCGAGACACTACTACGGTAATGAC GACGACACTGATTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 85 NI—302.52C9- VK GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGC CGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACGTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTC ATCTATTTGGGTTCTACTCGGGCCTCCGGGGTCCCTGACAGATTCAGTGGCA GTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGG ATGTTGGGGTTTATTACTGCTTACAAGCTCAACAAATTCCGTGGACGTTCGG CCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 87 NI-302.46C9- VH CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTAAAGCCTTCACAGACC CTGTCCCTCACCTGCACTGTTTCTGGTGCCTCCGTCAGCAGTGGTGCCTACTA CTGGAGTTGGATCCGGCAGCCCGCCGGGAAGCGACTGGAGTGGATTGGGCG TCCCACTTGGAGCACCAACTACAACCCCTCCCTCGAGAGTCGAGTC ACCATATCGTTAGACACGTCCAACAACCAGTTCTCCCTGAAGCTGACCTCTT TGACTGCCGCAGACACGGCCGTTTATTACTGTGCGAGAGAGGCTCCTGGTGA CTACGATGCTGCGCCCCTAGCCTACTGGGGCCAGGGCACCCTGGTCACCGTC TCCTCG SEQ ID NO: 89 NI-302.46C9- VK GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACA GAGTCACCATCACTTGCCGGGCAAGTCAGTACATTAGCCACTATTTAAATTG GTATCGGCAGAAACCAGGGAAAGCCCCTCAGCTCGTAATCTATGCTGCATCC AGTTTGCAAAGTGAGGTCCCATCAAGGTTCAGTGGGAGTGGATCTGGGCCA ACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATT ACTGTCAACAGAGTTACACTACCCCTCGAACTTTTGGCCAGGGGACCAAGCT GGAGATCAAA SEQ ID NO: 91 Table III: Nucleotide sequences of the VH and VL region of dies izing an epitope of the P-rich region of HTT, z'.e exon 1 in aggregated form.
Antibody Nucleotide sequences of variable heavy (VH) and variable light (VL) chains NI—302.63F3- VH CAGGTGCAGCTGGTGCAATCTGGGTCTGCGTTCAAGAAGCCTGGGACCTCA GTGAAAGTTTCCTGCAAGGCCTCTGGATACACCTTCGAGACCCGTTCTATGA ACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATACATGGGATGGATCA ACACCAACACTGGCAACCGCACGTATGTCCAGGCCTTCAGAGGACGATTTGT CTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAACTTA AAGACTGAGGACACTGCCGTGTATTACTGTGCGAGAGGGGCAGGTGGGGGA TATTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 5 NI-302.63F3—VK GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGA GGGCCACCATCAACTGCAAGTCCAATCAGAGTCTTTTCTACAGTTCCAACAA TAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCTG CTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGTG GCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTGA GGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTTG GCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 7 NI—302.31F11- VH GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCTTGATCCAGCCGGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGCAGCACCTACATGA GTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTTGAGTGCGTCTCAGTTATTTT TAGTGGCGCTGACACATATTACGCAGACTCCGTGAAGGGCCGATTCACCGTC TCCAGAGACAATTCCAAGAACACACTGTTTCTTCAGATGAACAGCCTGAGA GACACGGCCACATATTACTGTGTGAGACATTATTATGGTTCAGACC TTCCATCTGACTTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 13 NI-302.31F1 l- VK GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCGCCCCTGGAGAGC CGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTATACAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGAAGCCTCCACAGCTCCTG GTCTATTTGGGTTCTGATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCAAAGATTTTACACTGAACATCAGCAGAGTGGAGGCTGAGG ATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCGTGGACGTTCGG CCAAGGGACCAAGCTGGAGATCAAA SEQ ID NO: 15 NI-302.2A2— VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTACCTATTGGATGA TCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAA AACCAGATGGAAGTGACAAATACTATGTGGACTCTGTGAAGGGCCGATTCA CCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCC TGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGACGGCAGTG GCTGGAACGTCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 17 NI—302.2A2- VK GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGA CCATCAACTGCAAGTCCAGCCAGAGTCTTTTATACACCTCCAAAAA TAAGGACAGTAAGAACTACTTAGGTTGGTACCAGCAGAAACCAGGACAGCC GCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGAC CGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCC TGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAGTATTATACTACTCC TCAGTTCGGCGGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 19 NI-302.15D3- VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTTCAGCCTGGGGGGTCC CTAAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGC TCCGCCAAGCTCCAGGGAAGGGTCTGGTGTGGGTCTCACGTATTAG TAATGATGGCAGTAGCAAAACCTACGCGGACTCCGTGAAGGGCCGATTCAC CATCTCCAGAGACAACGCCAAAAACACGCTGTATCTGCAAATGAACAGTCT GAGAGCCGAGGACACGGCTGTGTATTACTGTGCAATACTTGGCGGATATTGT AGTAGTACCAGTTGTCGTCCCTTTGACAACTGGGGCCAGGGAACCCTGGTCA CCGTCTCCTCG SEQ ID NO: 135 NI—302.15D3- VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGA TCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGTTTATAACTATGT CTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTTTGAT GTCAGTAATCGGCCCTCAGGGATTTCTAATCGCTTCTCTGGCTCCAAGTCTG GCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTG ATTATTACTGCAGCTCATATACAAGCAGCGACACTTGGGTGTTCGGCGGAGG GACCAAGCTGACCATCCTA SEQ ID NO: 137 NI-302.64E5- VH GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCC CTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGACCAGGCCTGGATGA GCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGGATTA AAACGAAAACTGAGGGTGAAGCAACAGACTACGCAGCGCCCGTGAGAGGC AGATTCACCATCTCAAGAGATGATTCAGAAGACACGGTGTTTCTGCAAATGA ACAGCCTGAAAACCGAGGACACAGCCCTGTATTACTGTACGTCAACGGGAG TCTTAGCAGCAGCTGTCGATGTCTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCG SEQ ID NO: 164 NI-302.64E5- VK GACATCCAGTTGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGA GGGCCACCATGACCTGCAAGTCCAGCCAGAGTCTTTTCTACAGTTACAACAA TGAGAACTACTTAGCCTGGTATCAGCAGAGACCAGGACAGCCTCCTAAGTT GCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGT GGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTG AAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCAGACGTT CGGCCAAGGGACCAAAGTGGATATCAAA SEQ ID NO: 168 Table IV: Nucleotide sequences of the VH and VL region of antibodies recognizing an epitope of the C-terminal region of HTT, i.e exon 1 in aggregated form.
Antibody Nucleotide sequences of variable heavy (VH) and variable light (VL) chains NI-302.35C1-VH GAGGTGCAGCTGGTGGAGTCTGGGGGAAACTTGGTACAGCCGGGGGGGTCC CTGAGACTCTCCTGTACTGCCTCTGGATTCACCTTTAGTATAACGGCCCTGA TCCGCCAGGCTCCAGAAAAGGGGCCGCAGTGGGTCTCAGCAATCA CTGGAAATGCTTATGGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCA CCATTTCCAGAGACAACGCCAAGAACACACTGTACTTGCAAATGAACGGCC CCGAGGACACGGCCATCTATTACTGTGTGAAAGGAATTGCCTCCG ATAGTAGTGGTTATTCTGCCTTCTGGGGCCCGGGCACCCTGGTCACCGTCTC CTCG SEQ ID NO: 9 NI-302.35C1-VK GTGCTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAA GAGCCACCCTCTCCTGCAGGGCCAGTCAAAGTGTTGACAACCAGTTTGCCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATTTATGATGCATCC AGGAGGGCCCCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACA GACTTCACTCTCACCATTAGCAGCCTAGAGCCTGAAGATTTCGCAATTTATT ACTGTCAGCATCGTTACACCTGGCTCTACACTTTTGGCCAGGGGACACGACT GGAGATTAAA SEQ ID NO: 11 NI-302.72F10- VH GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTCGTACAGCCTGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGCAGTTATGCCATGA GCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTGTCAGATATCA GTGGTATTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCGTTTCAC CATTTCCAGAGACAATTCCGACAATACGTTGTACCTGGACATGAGCAGCCTG AGAGCCGAGGACACGGCCAGATATTACTGTGCGAAGGATCGAAAGCGCAGT TACGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 176 .72F10- VK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTACAGCCTGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGCAGTTATGCCATGA GCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTGTCAGATATCA GTGGTATTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCGTTTCAC CATTTCCAGAGACAATTCCGACAATACGTTGTACCTGGACATGAGCAGCCTG AGAGCCGAGGACACGGCCAGATATTACTGTGCGAAGGATCGAAAGCGCAGT GGCTGGTACGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 178 Table V: Nucleotide sequences of the VH and VL region of antibodies recognizing HTT species and/or fragments thereof.
Antibody Nucleotide sequences ofvariable heavy (VH) and variable light (VL) chains NI—302.6N9- VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTGCAGCCTGGGGGGTCC CTGAGACTCTCCTGTGTAGTCTCTGGATTCACCTTTAGTAGTTATGCCATGAC CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGCCTGGGTCTCAACAATTAG TGCTACTGGTGGTAGTACATTCTACACAGACTCCGTGAGGGGCCGGTTCACC ATCTCCCGAGACAATTCCAAGAACACACTGTATCTGCAAATGAATAGCCTGA GAACCGACGACACGGCCATATATTATTGTGTGAAAGATCTATTTGGAGTGGA CACCTCCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCG SEQ ID NO: 21 NI—302.6N9— VK GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA GAGCCACCCTCTCCTGCAGGCCCAGTCAGAGTGTCAGCGGCAGGTATGTGG CCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCTTCTATGCTGC ATCCAACAGGGCCATTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG CTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTG TGTCAGCACTATGGTGCCTCATCGTACACTTTTGGCCCGGGGACCA ATATCAAA SEQ ID NO: 23 NI-302.8F1— VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGAAGCCGGGGGGGTCC CTTACAATCTCCTGTGCAGCCTCTGGTTTCACCTTCAGTAATGCCTGGATGAA CTGGGTCCGCCAGGCTCCAGGTAAGGGGCTGGAGTGGGTCGGCCATATTAG AGCTGAAGGAGGGACATCAGACTATGCTGCACCCGTGAAAGGCAG ATTCACCATCTCAAGAGATGACTCAAAAAACACGCTGTATCTGCAAATGAA CAGCCTGAAAACCGAGGACACAGCCGTATATTATTGTATCCCCCCCCCCTAC TACTACTATTACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CCTCG SEQ ID NO: 81 NI-302.8F1— VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGA TCACCATCTCCTGCACTGGAGCCAGCAGTGATGTTGGGACTTATGACCTTGT CTCCTGGTACCAACAACATCCAGGCAAAGCCCCCAAACTCATTATTTATGAG GTCAATAAGCGGCCCTCAGGGGTTTCTTATCGCTTCTCTGCCTCCAAGTCTGC CAACACGGCCTCCCTGACAATATCTGGGCTCCAGGCTGAGGACGAGGCTGA ATATTACTGCTGCTCATATGCAGGTTATAGCACGGTATTCGGCGGAGGGACC AAGCTGACCGTCCTA SEQ ID NO: 83 NI—302.4A6— VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGCTTATGCCATGA GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTA GTGGTAGTGGTGGTAGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCTC CATCTCCAGAGACAACTCCAAAAACACCCTGTATCTGCAAATGAACAGCCT GAGAGCCGAGGACACGGCCGTATATTTCTGTGCGAAAGTTACCACGGAACT CTACGGTGCTAACTCCTACTACTACTACATGGACGTCTGGGGCAAAGGGACC ACGGTCACCGTCTCCTCG SEQ ID NO: 184 NI-302.4A6— VK GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA CCCTCTCCTGCAGGGCCAGTCAGAGTGTTGTCAGCAGGTATTTAGC CTGGTACCAGCAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGG ACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAATGT ATTACTGTCAGCTGTATGGTAACTCACAGACGTTCGGCCAGGGGACCAAGGT GGAGATCAAA SEQ ID NO: 186 NI-302.12H2- VH GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC CTGAGACTTTCCTGTGAAGCCTCTGGATTCACCTTTAGCAACTATGCCATGG GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTAATTA GTGGTACTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCA CCATCTCCAGAGACAATTCCATGAACACGCTGTATCTGCAAATGAACAGCCC GAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATCTGAGGAAGAT TAGCGGTCCTTTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTCG SEQ ID NO: 188 .12H2- VK GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC CTGAGACTTTCCTGTGAAGCCTCTGGATTCACCTTTAGCAACTATGCCATGG GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTAATTA GTGGTACTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCA CCATCTCCAGAGACAATTCCATGAACACGCTGTATCTGCAAATGAACAGCCC GAGAGCCGACGACACGGCCGTATATTACTGTGCGAAAGATCTGAGGAAGAT TAGCGGTCCTTTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTCG SEQ ID NO: 192 NI-302.8M1— VH GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCA GTGAAAGTTTCCTGCAAGGCATCCGGATACACCTTCACCATCTACTATATGC TGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGAATCA GCCCGAGTGGTGCCCACACAATGTACGCACAGAATTTCCAGGGCAGAGTCA CCGTGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGAGCACGGTGA CTAACTATCGACCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCG SEQ ID NO: 194 NI-302.8M1- VK GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACA GAGTCACTATCACTTGCCGGGCGAGTCAGGACATTAGCAATTATTTAGCCTG GCAGAAACCAGGGAAAGTTCCTAAACTCCTGATCTTTGCTGCATCC ACTTTGCAATCAGGGGTCCCGTCTCGGTTCGGTGGCAGTGGATCTGGGACAG ATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTA CTGTCAAAACTATAACAGTGGCCCTCCGCCTTTCGGCCCTGGGACCAAAGTG GATATCAAA SEQ ID NO: 198 Table VI: Nucleotide sequences ofthe VH and VL region of antibodies an epitope of the N- terminal-region of HTT, z'.e exon 1 in ated form Antibody Nucleotide sequences ofvariable heavy (VH) and variable light WL) chains NI-302.15E8- VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATACAGCCGGGGGGGTCC CTGAGACTCTCCTGTGCAGTCTCTGGATTCACCGTCAGTAGTTATAGCATGA ACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATACACTA GTAGTAGCAGAAGTAATACCAAAAAGTACGCAGACTCTGTGAAGGGCCGAT TCACCATCTCTAGAGACAATGCCAGGAACTCACTCTATCTGCAAATGAACAG CCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGCAGGGGACTT CGGGGAGTTACTCACTGGTGAGGGGTATTACGGTATGGACGTCTGGGGCCA AGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 131 NI-302.15E8- VL GAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAG CCACCATCACCTGCTCGGGAGATGAATTGGGGGATAAATATGTTGGTTGGTA TCAACAGAAGCCAGGCCAGTCCCCTCTGCTGGTCATCTATCAAGATGCGAAG CGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAG CCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTACTACTG TCAGGCGTGGGACAGCGGCACGATGGTTTTCGGCGGAGGGACCAGGCTGAC CGTCCTA SEQ ID NO: 133 Table VII: Nucleotide sequences of the VH and VL region of antibodies recognizing an epitope of the Q/P-rich region of HTT, z'.e exon 1 in aggregated form.
Antibody Nucleotide sequences ofvariable heavy (VH) and variable light (VL) chains NI—302.7D8- VH CAGGTGCAGCTGGTGCAATCTGGATCTGAGTTGAAGAAGCCTGGGGCCTCA GTGAAGGTTTCCTGCAAGGCTTCTGGATACAACTTCAATAACTATGCCATCA ATTGGTTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCA ACACCATCACTGGGCACCCAACGTATGCCCAGGGCTTCAAAGGACGATTTGT CTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAGCCTA AAGCCTGAGGACACTGCCGTCTATTACTGTGCGAGAACTTACAGTAACTACG GCGAATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 172 .7D8— VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCGTGGACAGTCGA TCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGAAGTTATAACCTTGT CTCCTGGTACCAACAGTACCCAGGCAAGGCCCCCAAGCTCATAATTCATGAG GGCAGTGAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTG GCAACACGGCCTCCCTGACAATTTCTGGGCTCCAGGCTGAGGACGAGGCTG ATTATTACTGCTGCTCATATGCAGGTACTACTACTTTCGTGCTATTCGGCGGA GGGACCAAGCTGACCGTCCTC SEQ ID NO: 174 Due to the cloning strategy the amino acid sequence at the N- and C-terminus of the heavy chain and light chains may potentially contain -induced alterations in FRl and FR4, which however do not ntially affect the biological activity of the antibody. In order to provide a consensus human antibody, the nucleotide and amino acid sequences of the original clone can be aligned with and tuned in accordance with the pertinent human germ line variable region sequences in the database; see, e.g., Vbase2, as described above. The amino acid sequence of human antibodies are indicated in bold when N- and C-terminus amino acids are ered to potentially deviate from the consensus germ line ce due to the PCR primer and thus have been replaced by primer-induced on correction , see Table VI.
Accordingly, in one ment of the present invention, the polynucleotide comprises, consists ially of, or consists of a nucleic acid having a polynucleotide sequence of the VH and the VL region of an anti-HTT antibody and/or fragments thereof as depicted in Table VI.
Table VIII: Nucleotide sequences of the VH and VL region of antibodies recognizing HTT species and/or fragments thereof showing replacement by PIMC (bold).
Alternative Nucleotide sequences ofvariable heavy WH) and variable light WL) chains Antibody-regions with PIMC Nl-302.33Cl l-PIMC CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGGGAACTC VH ACTCTCCTGTGCAGCGTCTGGATTCAGGTTCAGTGACTTTGGCATG CACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGCTGGCACTTATA TGGTATGATGGAGGGTATAAGTACTATGCAGACTCCGTGAAGGGCCGATTC TCCAGAGACAATTCCAAGAATACGATGTTTCTACAAATGAACAGCC TGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGACCCACCTAGAATATTG CAGTAGAACCACCTGCTATCTCGGCCACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCG SEQ ID NO: 97 NI-302.33C11-PIMC GACATCCAGTTGACCCAGTCTCCGTCCTTCCTATCTGCGTCTGTGGGAGAC VK ACAGTCACCTTCACTTGCCGGGCCAGTCAGGGCATTAGCGATTATTTAGCCT GGTTTCAGCAGAAACCAGGGATTGCCCCTAAGCTCCTGATCTATGCTGCGTC CACTTTGCAAACCGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC AGAATTCACTCTCACAATCCGCAGCCTGCAGTCTGAAGATTTTGGAACTTAT TACTGTCAGCAGCTTAAAACTTACCCGTACACTTTTGGCCAGGGGACCAAG CTGGAGATCAAA SEQ ID NO: 99 NI-302.63F3-PIMC GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAG VK AGGGCCACCATCAACTGCAAGTCCAATCAGAGTCTTTTCTACAGTTCCAACA ATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCT GCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGT GGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTG TTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 101 NI-302.63F3-PIMC- GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAG NS VK AGGGCCACCATCAACTGCAAGTCCTCACAGAGTCTTTTCTACAGTTCCAACA ACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCT GCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGT GGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTG AGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 103 .63F3-PIMC- GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAG SG VK AGGGCCACCATCAACTGCAAGTCCAATCAGGGCCTTTTCTACAGTTCCAACA ATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCT GCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGT GGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTG AGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 105 NI—302.63F3-PIMC— GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAG NQ VK AGGGCCACCATCAACTGCAAGTCCCAACAGAGTCTTTTCTACAGTTCCAACA ATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCT GCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGT GGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTG AGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTT GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 107 NI-302.35C1-PIMC GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAA VK AGAGCCACCCTCTCCTGCAGGGCCAGTCAAAGTGTTGACAACCAGTTTGCCT GGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATTTATGATGCATC CAGGAGGGCCCCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGAC AGACTTCACTCTCACCATTAGCAGCCTAGAGCCTGAAGATTTCGCAATTTAT TACTGTCAGCATCGTTACACCTGGCTCTACACTTTTGGCCAGGGGACCAAG CTGGAGATCAAA SEQ ID NO: 109 NI—302.3 1F] 1-PIMC GATATTGTGATGACTCAATCACCACTCTCCCTGCCCGTCGCCCCTGGAGAG VK CCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTATACAGTAATGGAT ATTTGGATTGGTACCTGCAGAAGCCAGGGAAGCCTCCACAGCTCCT GGTCTATTTGGGTTCTGATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGC TCAGGCAAAGATTTTACACTGAACATCAGCAGAGTGGAGGCTGAG GATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCGTGGACGTTCG GCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 111 NI-302.2A2—PIMC GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAG VK AGGGCCACCATCAACTGCAAGTCCAGCCAGAGTCTTTTATACACCTCCAAAA ATAAGGACAGTAAGAACTACTTAGGTTGGTACCAGCAGAAACCAGGACAGC CTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGA CCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGC CTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAGTATTATACTACTC CTCAGTTCGGCGGAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 1 13 NI-302.74C11—PIMC CAGGTGCAGCTGGTGCAATCTGGGACTGAGGTGCAGAAGCCTGGGGCCTC VH AGTAAAAGTCTCCTGCAAGGCTTCTGGATACAGTTTCACCGGCTACTTTTTG CACTGGGTACGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGTGGATC AACCCTAACAGTGGTGACACAAACTATGCAGAGAAGTTTCGGGGCAGAATC ATCATGACCAGGGACACGTCTGTCAGCACAGCCCACATGGAGTTGAGCAGC CTGAGATTTGACGACACGGCCCTATATTACTGTACGAGAGAGGCCCCTGACC CGGGCGCTGAGACGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCC SEQ ID NO: 1 15 .74C11-PIMC TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGAC VL GGCCAGGATCACCTGCTCTGGAGATGCAGTGCCAAAGCAGTATATTTATTGG TACCAGCAGAAGCCAGGCCAGGCCCCTATTCTGGTGATATATAAAGACACT CCTTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCAGGGACAA CAGTCACGTTGACCATAACTGGCGTCCAGGCAGACGACGAGGGTGACTATT ACTGTCAATCAGCAGACAGTAGTGCTACTTGGGTGTTCGGCGGAGGGACCA AATTGACCGTCCTA SEQ ID NO: 117 NI—302.39G12-PIMC GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCACCCTTGGGGGTC VH CCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTAATTACGCCATA ACTTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGCAATATATTTCAGTAATTT ATCGTGATGGCAGGACATACTACGGAGACTCCGTGAGGGGCCGCTTCACCA TCTCTAGGGACGATTCCAAGAACACTCTCTATCTTCAAATGAACAGCCTGAG GGACACGGCTGTGTATTACTGTGCGAGAGCGCACGGCCAATATTAC TATGGTGTGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 119 NI-302.39G12-PIMC GACATCGTGATGACCCAGTCTCCACTCTCCCTGTCCGTCAGCCCTGGAGAG VK CCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGAT ACAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAGCTCCT GATCTATTTGAGTTCTAATCGGCCCTCCGGGGTCCCTGATAGGTTCAGTGCC AGTGGATCAGGCACAGAGTTCACACTGCAAATCAGCAGAGTGGAGGCTGAG GATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCG GAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 121 NI-302.11A4-PIMC GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGAGAA VK AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTCG ACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTAC GTCCCGCAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGG GACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTG TATTACTGTCAACAGTATGGTAGCTCGTGGACGTTCGGCCCAGGGACCAAG GTGGAAATCAAA SEQ ID NO: 123 NI-302.22H9—PIMC GATATTGTGATGACTCAATCACCACTCTCCCTGTCCGTCAGCCCTGGAGAG VK CCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGAT ACAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAACTCCT GATCTATTTGAATTCTAATCGGGCCTCCGGGGTCCCTGATAGGTTCAGTGGC AGTGGATCAGGCACAGAGTTCACACTGACAATCAGCAGAGTGGAGGCTGAG GATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCG GAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 125 NI-302.44D7-PIMC CAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGT VH CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCAT GAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTAT TGGTTATAGTGATACTAGCACATATTACGCAGACTCCGTGAAGGGCCGCTTC ACCGTCTCCAGAGACATTTCCAAGAACACGCTGTATCTGCAAATGAATAGCC TGAGGGCCGAGGACACGGCCGTATATTACTGCGCGAAAGGTACCAGGGACT ATTACGGTATGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 127 NI-302.78H12-PIMC CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA VH CCCTGTCCCTCACCTGTCTTGTCTCTAGTTACTCCATCAGCAATGGTTACTAC TGGGGCTGGATTCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGT ATCTATCATAATGGGAACACCTATTACAACCCGTCCCTCAAGAGTCGAGTCA TCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGTTGAGGTCTGT GACCGCCGCAGACACGGCCGTGTACTACTGTGCGATGCCAAGTGCCACCTAT TATTATGGTTCGGGGACTCAATTCCATGCGTTTGATGTCTGGGGCCAAGGGA CAATGGTCACCGTCTCTTCG SEQ ID NO: 129 NI-302.64E5—PIMC GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGT CCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGACCAGGCCTGGAT VH GAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGGAT TAAAACGAAAACTGAGGGTGAAGCAACAGACTACGCAGCGCCCGTGAGAG GCAGATTCACCATCTCAAGAGATGATTCAGAAGACACGGTGTTTCTGCAAAT GAACAGCCTGAAAACCGAGGACACAGCCCTGTATTACTGTACGTCAACGGG AGTCTTAGCAGCAGCTGTCGATGTCTACTGGGGCCAGGGCACCCTGGTCAC CGTCTCCTCG SEQ ID NO: 166 NI-302.64E5-PIMC GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAG ACCATGACCTGCAAGTCCAGCCAGAGTCTTTTCTACAGTTACAACA VK ATGAGAACTACTTAGCCTGGTATCAGCAGAGACCAGGACAGCCTCCTAAGT TGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAG TGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCT GAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCAGACGT TCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 170 NI—302.72F10-PIMC GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGCAGTTATGCCAT VH GAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTGTCAGATAT CAGTGGTATTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCGTTTC ACCATTTCCAGAGACAATTCCGACAATACGTTGTACCTGGACATGAGCAGCC TGAGAGCCGAGGACACGGCCAGATATTACTGTGCGAAGGATCGAAAGCGCA GTGGCTGGTACGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 178 .72F10-PIMC GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGACTTTGTCTCCAGGGGAA ACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCGCCTACTTAGGCT VK GGTATCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATC CATTAGGGCCACTGGCATTCCAGACAGGTTTAGTGGCAGTGGGTCTGGGAC AGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTCTGCAGTTTAT TACTGTCACCAGCGTAGCAAGTGGCCTCTTACTTTCGGCGGAGGGACCAAG GTGGAAATCAAA SEQ ID NO: 182 .12H2-PIMC GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTTTCCTGTGAAGCCTCTGGATTCACCTTTAGCAACTATGCCAT VH GGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTAAT TAGTGGTACTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTC ACCATCTCCAGAGACAATTCCATGAACACGCTGTATCTGCAAATGAACAGCC CGAGAGCCGACGACACGGCCGTATATTACTGTGCGAAAGATCTGAGGAAGA GTCCTTTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCA CGGTCACCGTCTCCTCG SEQ ID NO: 190 NI—302.8M1-PIMC CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC AGTGAAAGTTTCCTGCAAGGCATCCGGATACACCTTCACCATCTACTATATG VH CACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGAATC AGCCCGAGTGGTGCCCACACAATGTACGCACAGAATTTCCAGGGCAGAGTC ACCGTGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGC CTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGAGCACGGTG ACTAACTATCGACCCTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTC TCCTCG SEQ ID NO: 196 The present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention.
The polynucleotides may be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides, e.g., as described in Kutmeier et al., BioTechniques 17 (1994), 242, which, , involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, ing and ng of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof may be generated from nucleic acid from a suitable source. If a clone containing a c acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be ally synthesized or obtained from a suitable source (e.g, an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+ RNA, isolated from, any tissue or cells expressing the HTT-specific antibody, such as oma cells selected to express an antibody) by PCR cation using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by g using an oligonucleotide probe specific for the ular gene sequence to identify, e. g., a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be cloned into able cloning vectors using any method well known in the art.
Once the nucleotide ce and corresponding amino acid sequence of the antibody, or antigen-binding fragment, variant, or derivative thereof is determined, its tide sequence may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site ed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1990) and Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), which are both orated by nce herein in their entireties), to generate antibodies having a ent amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
IV. Expression of Antibody Polypeptides ing manipulation of the isolated genetic material to provide antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention, the polynucleotides encoding the antibodies are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of antibody. Recombinant expression of an antibody, or fragment, derivative, or analog thereof, e. g., a heavy or light chain of an dy which binds to a target molecule is described herein. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain le domain), of the invention has been ed, the vector for the production of the antibody le may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding tide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational l signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in viva genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide ce encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operable linked to a er. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., international applications WO 86/05 807 and WO 89/01036; and US patent no. 5,122,464) and the le domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The term "vector" or "expression vector" is used herein to mean vectors used in accordance with the present invention as a e for introducing into and expressing a desired gene in a host cell. As known to those d in the art, such vectors may easily be selected from the group consisting of plasmids, phages, s, and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, riate restriction sites to facilitate g of the desired gene and the ability to enter and/0r replicate in eukaryotic or yotic cells. For the purposes of this invention, numerous expression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, iruses (RSV, MMTV or , or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more s which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, e resistance (e.g, antibiotics), or resistance to heavy metals such as . The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. onal elements may also be needed for optimal synthesis of mRNA. These elements may e signal ces, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
In particularly preferred embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) as discussed above. In one embodiment, this is accomplished using a proprietary expression vector of Biogen IDEC, Inc., referred to as NEOSPLA, and disclosed in US patent no. 730. This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, in phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene, and leader ce. This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate cation. Of course, any sion vector which is capable of eliciting expression in eukaryotic cells may be used in the present invention. Examples of suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.l, pEFl/His, pIND/GS, pRc/HCMV2, Zeo2, pTRACER—HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers oftransformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in US patent nos. 5,736,137 and 5,658,570, each of which is orated by reference in its entirety herein.
This system provides for high expression levels, e.g, > 30 l/day. Other exemplary vector systems are disclosed e.g., in US patent no. 6,413,777.
In other preferred embodiments the antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be expressed using polycistronic constructs such as those disclosed in US patent application publication no. 2003-0157641 A1 and incorporated herein in its ty. In these expression systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of antibodies. Compatible IRES sequences are disclosed in US patent no. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression s may be used to ively produce the fill range of antibodies disclosed in the instant application. Therefore, in one embodiment the present invention es a vector comprising the polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody, optionally in combination with a polynucleotide that encodes the variable region of the other immunoglobulin chain of said g molecule.
More generally, once the vector or DNA sequence encoding a monomeric subunit of the antibody has been prepared, the expression vector may be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by s techniques well known to those of skill in the art. These include, but are not limited to, transfection including lipotransfection using, e.g., Fugene® or lipofectamine, protoplast fiJsion, calcium phosphate precipitation, cell fusion with ped DNA, microinjection, and ion with intact virus. Typically, plasmid introduction into the host is via standard calcium phosphate co- precipitation method. The host cells harboring the sion construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. ary assay ques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence—activated cell sorter is (FAC S), histochemistry and the like.
The expression vector is transferred to a host cell by conventional techniques and the ected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein. Thus, the invention includes host cells comprising a polynucleotide encoding an dy of the invention, or a heavy or light chain thereof, or at least the binding domain or variable region of an immunoglobulin thereof, which preferably are operable linked to a heterologous promoter. In addition or alternatively the invention also includes host cells comprising a vector, as defined hereinabove, comprising a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody, optionally in combination with a polynucleotide that encodes the variable region of the other immunoglobulin chain of said binding molecule. In preferred embodiments for the expression of double-chained antibodies, a single vector or vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as ed below.
The host cell may be nsfected with two sion vectors ofthe invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain d polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
As used herein, "host cells" refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions ofprocesses for isolation of antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source body unless it is clearly ed otherwise. In other words, recovery ofpolypeptide from the "cells" may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
A variety of host-expression vector systems may be utilized to express antibody molecules for use in the methods described herein. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding ces, express an dy molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis) ormed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression s containing antibody coding ces; insect cell systems infected with recombinant virus expression s (e.g., baculovirus) containing antibody coding sequences; plant cell s infected with recombinant virus expression vectors (e.g, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid sion vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g, COS, CHO, NSO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from ian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as E. coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells, in conjunction with a vector such as the major ediate early gene promoter element from human cytomegalovirus is an ive expression system for antibodies; see, e. g., ng et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 , 2.
The host cell line used for protein expression is often ofmammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CH0 (Chinese r Ovary), DG44 and DUXBll (Chinese Hamster Ovary lines, DHFR minus), HELA (human al carcinoma), CVI (monkey kidney line), COS (a derivative of CV1 with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, W138, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), Ag3.653 (mouse a), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). CH0 and 293 cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
In on, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g, glycosylation) and processing (e.g., cleavage) of protein products may be important for the fiinction of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host s can be chosen to ensure the correct modification and processing ofthe foreign n expressed. To this end, eukaryotic host cells which possess the cellular ery for proper sing of the primary transcript, glycosylation, and orylation of the gene product may be used.
For long-term, high-yield production of recombinant proteins, stable expression is preferred.
For e, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control ts (e.g., promoter, er, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably ate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which stably s the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine oribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc.
Natl. Acad. Sci. USA 78 (1981), 2072); neo, which s resistance to the aminoglycoside G-418 Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. l. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191- 217; TIB TECH 11 (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147. Methods ly known in the art of inant DNA technology which can be used are described in Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A tory , Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli er al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification, for a ; see Bebbington and Hentschel, The use of vectors based on gene cation for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor t in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also se; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.
In vitro production allows scale-up to give large s of the desired polypeptides.
Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography s, for example gel filtration, ion-exchange chromatography, chromatography over ellulose or o-) affinity chromatography, e. g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
Genes encoding antibodies, or antigen-binding fragments, variants or derivatives thereofofthe invention can also be expressed in non-mammalian cells such as bacteria or insect or yeast or plant cells. ia which readily take up nucleic acids include members of the enterobacteriaceae, such as s of E. coli or ella; Bacillaceae, such as B. is; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into fimctional molecules. Where tetravalent forms of antibodies are desired, the ts will then self-assemble into tetravalent antibodies; see, e.g., international application W0 948.
In bacterial systems, a number ession vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of filSlOIl n products that are y purified may be desirable. Such vectors include, but are not d, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2 (1983), 1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke and Schuster, J. Biol. Chem. 24 (1989), 5503- 5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix of hione- e beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to e thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number ofother strains are commonly available, e.g., Pichia pastoris. For sion in romyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature 282 (1979), 39; Kingsman et (1]., Gene 7 (1979), 141; Tschemper er al., Gene 10 (1980), 157) is ly used. This d already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
In an insect system, Autographa calz‘form’ca nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugz'perda cells. The dy coding sequence may be cloned individually into non-essential regions (for example the drin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
Once an antibody molecule of the invention has been recombinantly sed, the whole antibodies, their dimers, individual light and heavy chains, or other globulin forms of the present invention, can be purified ing to standard procedures of the art, including for example, by chromatography (e.g, ion exchange, affinity, particularly by affinity for the c antigen after Protein A, and sizing column chromatography), centrifiigation, differential solubility, e. g. ammonium sulfate precipitation, or by any other standard technique for the purification of proteins; see, e.g., Scopes, "Protein ation", Springer Verlag, NY. (1982). Alternatively, a preferred method for sing the affinity of antibodies of the invention is disclosed in US patent publication 2002-0123057 A1. In one embodiment therefore, the present invention also provides a method for ing an anti-HTT antibody or an antibody recognizing mutated and/or aggregated HTT species and/or fragments thereof or immunoglobulin chain(s) thereof, said method comprising: (a) culturing the host cell as defined above, which cell comprised a polynucleotide or a vector as defined hereinbefore; and (b) isolating said antibody or immunoglobulin chain(s) thereof from the culture.
Furthermore, in one embodiment the present invention also relates to an antibody or immunoglobulin s) thereof encoded by a polynucleotide as defined above or obtainable by said method for preparing an anti-HTT antibody or an dy recognizing mutated and/or aggregated HTT species and/or fragments thereof or immunoglobulin chain(s) V. Fusion Proteins and Conjugates In n embodiments, the antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, a single-chain Fv antibody fragment of the ion may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label such as a fluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and the like) An antibody polypeptide of the invention may se, consist essentially of, or consist of a filSlOIl protein. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin HTT-binding domain with at least one target binding site, and at least one heterologous portion, z'.e., a n with which it is not naturally linked in nature. The amino acid sequences may normally exist in te proteins that are brought together in the fusion polypeptide or they may normally exist in the same n but are placed in a new arrangement in the filSlOIl polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
The term "heterologous" as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared. For instance, as used , a "heterologous polypeptide" to be fiised to an antibody, or an antigen-binding fragment, variant, or analog thereof is d from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non- immunoglobulin polypeptide of a different species.
As discussed in more detail ere herein, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N— or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector les such as heterologous polypeptides, drugs, radionuclides, or toxins; see, e.g., international applications WO 92/08495; W0 91/14438; W0 89/12624; US patent no. ,314,995; and an patent application EP 0 396 387.
Antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, z'.e., peptide isosteres, and may n amino acids other than the 20 gene-encoded amino acids.
Antibodies may be d by l processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well bed in basic texts and in more ed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification may be present in the same or varying degrees at l sites in a given antibody. Also, a given antibody may contain many types of modifications. Antibodies may be branched, for example, as a result of ubiquitination, and they may be , with or without branching. Cyclic, ed, and branched cyclic antibodies may result from posttranslational natural processes or may be made by synthetic methods. ations include acetylation, ion, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent ment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, de bond formation, demethylation, formation of nt cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma—carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, er-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination; see, e.g., Proteins - Structure And Molecular Properties, T. E. Creighton, W. H.
Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification OfProteins, B. C. Johnson, Ed., Academic Press, New York, (1983) 1-12; Seifter et al., Meth.
Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).
The present invention also es for fusion proteins comprising an antibody, or antigen- binding fragment, t, or derivative thereof, and a heterologous polypeptide. In one embodiment, a fusion protein of the invention comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH s of an antibody of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or nts or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the VH-CDRs of an antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL- CDRs of an antibody, or fragments, variants, or derivatives f, and a heterologous polypeptide sequence. In one embodiment, the filSiOIl protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an antibody ofthe present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fiision protein specifically binds to HTT. In r ment, a fusion n comprises a polypeptide having the amino acid sequence of at least one VH region of an antibody of the invention and the amino acid sequence of at least one VL region of an antibody of the invention or nts, derivatives or variants thereof, and a heterologous polypeptide sequence. Preferably, the VH and VL regions of the fiJsion protein correspond to a single source antibody (or scFV or Fab fragment) which specifically binds HTT. In yet another embodiment, a fusion protein for use in the diagnostic and ent methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs of an antibody and the amino acid ce of any one, two, three, or more of the VL CDRs of an antibody, or nts or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six, or more of the (s) or VL-CDR(s) correspond to single source antibody (or scFV or Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
Exemplary fusion proteins reported in the literature include qulOI‘lS of the T cell or (Gascoigne er al., Proc. Natl. Acad. Sci. USA 84 , 940; CD4 (Capon er al., Nature 337 (1989), 525-531; Traunecker er al., Nature 339 (1989), 68-70; Zettrneissl et (11., DNA Cell Biol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990), 667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167); CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (1991),721-730); CTLA-4 (Lisley et al., J. Exp. Med. 174 (1991), 561- 569); CD22 (Stamenkovic er al., Cell 66 , 144); TNF or (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991); and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).
As discussed elsewhere herein, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be fused to heterologous polypeptides to increase the in viva half—life of the polypeptides or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to the antibodies of the ion to increase their half-life in viva; see, e. g., Leong et al., Cytokine 16 (2001), 106—119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.
Moreover, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a e to facilitate their purification or detection. In preferred embodiments, the marker amino acid ce is a hexa-histidine peptide (HIS), such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance, hexa- histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which ponds to an epitope derived from the influenza lutinin protein n et al., Cell 37 (1984), 767), GST, c- mycand the "flag" tag; see, e.g, Bill Brizzard, hniques 44 (2008) 693-695 for a review ofepitope tagging techniques, and Table 1 on page 694 therein listing the most common e tags usable in the present invention, the subject matter of which is hereby expressly incorporated by reference.
Fusion proteins can be prepared using methods that are well known in the art; see for e US patent nos. 5,116,964 and 5,225,538. The precise site at which the fusion is made may be selected empirically to optimize the secretion or g characteristics of the filSlOIl protein.
DNA encoding the fiision protein is then transfected into a host cell for sion, which is performed as described hereinbefore.
Antibodies ofthe t invention may be used in non-conjugated form or may be ated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient. Antibodies, or n-binding fragments, variants, or derivatives thereof of the invention can be labeled or conjugated either before or after purification, when ation is performed. In ular, antibodies, or antigen-binding fragments, ts, or derivatives thereof of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, Viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The antibodies of the present invention can be used in a corresponding way to obtain such toxins. Illustrative of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.
Those skilled in the art will iate that conjugates may also be assembled using a y oftechniques ing on the ed agent to be ated. For example, conjugates with biotin are prepared, e.g., by ng a HTT-binding polypeptide with an activated ester in such as the biotin N—hydroxysuccinimide ester. Similarly, conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g. those listed herein, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of the antibodies, or antigen-binding fragments, variants or derivatives thereof of the invention are prepared in an analogous manner.
The t invention further encompasses antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, demonstrate presence of a HTT amyloidosis to indicate the risk of getting a disease or disorder associated with d and/or aggregated HTT, to monitor the development or progression of such a disease, zle. a disease showing the occurrence of, or related to aggregated HTT, or as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. In one embodiment thus, the present ion relates to an antibody, which is detectably labeled.
Furthermore, in one embodiment, the present ion relates to an antibody, which is attached to a drug. Detection can be facilitated by ng the antibody, or antigen-binding fragment, variant, or derivative f to a detectable substance. The detectable substances or label may be in general an enzyme; a heavy metal, preferably gold; a dye, preferably a cent or luminescent dye; or a radioactive label. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive gnetic metal ions; see, e.g., US patent no. 4,741,900 for metal ions which can be ated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, alactosidase, or cholinesterase; es of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of inescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc. Therefore, in one embodiment the present ion provides a detectably labeled antibody, wherein the detectable label is selected from the group ting of an enzyme, a radioisotope, a fluorophore and a heavy metal.
An antibody, or antigen-binding fragment, variant, or derivative thereof also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of ularly useful chemiluminescent labeling compounds are l, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
One of the ways in which an antibody, or antigen-binding fragment, variant, or derivative thereof can be detectably labeled is by g the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Imrnunosorbent Assay (ELISA)" Microbiological Associates Quarterly ation, Walkersville, Md, Diagnostic Horizons 2 (1978), l-7); Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. l. 73 (1981), 482-523; Maggio, (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme, which is bound to the antibody, will react with an appropriate substrate, preferably a genic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not d to, malate ogenase, staphylococcal nuclease, deltasteroid ase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucosephosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be lished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic on of a substrate in comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other assays. For example, by radioactively labeling the dy, or antigen-binding nt, variant, or derivative thereof, it is possible to detect the antibody through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation r, or diography.
An antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled using cence emitting metals such as 152Eu, or others of the lanthanide series.
These metals can be attached to the antibody using such metal ing groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
Techniques for conjugating various moieties to an antibody, or antigen-binding fragment, t, or derivative thereof are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, ld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed), Robinson et al. (eds), Marcel Dekker, Inc., (1987) 623-53; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And al Applications, Pinchera et al. (eds), (1985) 475-506; "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in onal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds), Academic Press (1985) 303—16, and Thorpe er al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62 (1982), 119-158.
As mentioned, in certain embodiments, a moiety that enhances the stability or efficacy of a binding molecule, e.g, a binding polypeptide, e.g., an antibody or immunospecific nt thereof can be conjugated. For example, in one embodiment, PEG can be conjugated to the binding molecules ofthe invention to increase their half-life in viva. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug DeliV. Rev. 54 (2002), 531; or Weir et (1]., Biochem. Soc.
Transactions 30 , 512.
VI. Compositions and Methods of Use The present invention relates to compositions comprising the aforementioned HTT-binding molecule, e.g, antibody or antigen—binding fragment thereof of the present invention or tive or variant thereof, or the polynucleotide, vector or cell of the ion as defined hereinbefore. In one embodiment, the composition of the present invention is a ceutical composition and further comprises a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention may comprise further agents such as interleukins or interferons depending on the ed use of the pharmaceutical composition.
For use in the treatment ofa disease or disorder showing the occurrence of, or related to mutated and/or aggregated HTT, such as HTT amyloidosis, the additional agent may be selected from the group consisting of small organic les, anti-HTT antibodies, and combinations f. Hence, in a particular red embodiment the present invention relates to the use of the HTT-binding molecule, e.g., antibody or n-binding fragment thereof of the present invention or of a binding molecule having substantially the same binding specificities of any one thereof, the polynucleotide, the vector or the cell ofthe present invention for the preparation of a pharmaceutical or stic composition for prophylactic and eutic treatment of Huntington's disease (HD) and/or a disease or disorder associated with HTT and/or HTT amyloidosis, monitoring the progression of HD and/or a disease or disorder associated with HTT and/or HTT amyloidosis or a se to a HTT amyloidosis treatment in a subject or for determining a subj ect's risk for ping a disease or disorder associated with HTT.
Hence, in one embodiment the present invention s to a method of treating a disease or er characterized by abnormal accumulation and/or deposition of HTT and/or aggregated and/or mutated HTT in affected systems and organs which method comprises administering to a subject in need thereof a therapeutically ive amount of any one of the afore-described HTT-binding molecules, antibodies, polynucleotides, vectors or cells of the instant invention.
A particular advantage of the therapeutic approach of the t invention lies in the fact that the recombinant dies of the present invention are derived from B cells or memory B cells from healthy human subjects with no signs or symptoms of a e, e. g. ng an asymptomatic mutation and/or mutations, showing the occurrence of, or related to aggregated HTT and thus are, with a certain probability, capable of preventing a clinically st disease related to d and/or aggregated HTT, or of diminishing the risk of the ence of the clinically manifest e or disorder, or of delaying the onset or progression of the clinically manifest disease or disorder. Typically, the antibodies ofthe t invention also have already successfully gone through c maturation, z'. e. the optimization with respect to selectivity and effectiveness in the high affinity binding to the target HTT molecule by means of somatic variation of the variable regions of the antibody.
The knowledge that such cells in viva, e. g. in a human, have not been activated by means of related or other physiological proteins or cell structures in the sense of an autoimmunological or allergic reaction is also of great medical importance since this signifies a considerably increased chance of successfully living through the clinical test phases. So to speak, efficiency, acceptability and tolerability have already been demonstrated before the preclinical and al development of the prophylactic or therapeutic antibody in at least one human subject. It can thus be expected that the human-derived anti-HTT antibodies of the present invention, both its target structure-specific efficiency as therapeutic agent and its decreased probability of side effects significantly increase its clinical ility of success.
The present invention also provides a pharmaceutical and diagnostic, respectively, pack or kit comprising one or more containers filled with one or more of the above described ingredients, e. g. anti-HTT antibody, binding fragment, derivative or variant thereof, polynucleotide, vector or cell of the present invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition or alternatively the kit comprises reagents and/or instructions for use in appropriate diagnostic assays. The ition, e.g. kit of the present invention is of course particularly suitable for the risk ment, diagnosis, prevention and treatment of Huntington's e and/or a disease or disorder which is accompanied with the presence of mutated and/or aggregated HTT, and in particular applicable for the treatment of disorders generally characterized by HTT amyloidosis.
The pharmaceutical itions of the present invention can be formulated according to methods well known in the art; see for example ton: The Science and Practice of Pharmacy (2000) by the University ofSciences in Philadelphia, ISBN 0306472. Examples of suitable pharmaceutical rs are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile ons etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal stration or spinal or brain delivery. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. ations for rectal or vaginal administration may be presented as a itory with a suitable carrier.
The dosage regimen will be determined by the attending physician and al factors. As is well known in the medical arts, dosages for any one patient s upon many factors, including the patient‘s size, body e area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 ug (or eic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
Generally, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body . For example dosages can be 1 mgkg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, ably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple s over a prolonged period, for example, of at least six months. Additional exemplary treatment regimens entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal dies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Progress can be monitored by periodic assessment. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous ts are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl . Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline, and ed media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for example, antimicrobials, anti- ts, chelating agents, and inert gases, and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as dopamine or psychopharmacologic drugs, ing on the intended use of the pharmaceutical composition.
Furthermore, in a preferred embodiment of the present invention the pharmaceutical composition may be formulated as a vaccine, for e, if the pharmaceutical composition of the invention comprises an anti-HTT antibody or HTT-binding fragment, tive or synthetic or biotechnological variant f for passive immunization. As ned in the background section mutated and/or aggregated HTT s and/or fragments or derivatives thereof are a major trigger for HTT amyloidosis. Accordingly, it is prudent to expect that passive immunization with human anti-HTT dies and equivalent HTT-binding molecules ofthe t invention will help to circumvent several adverse s of active immunization therapy concepts and lead to a reduced aggregation of HTT. Therefore, the present anti-HTT dies and their equivalents of the present invention will be particularly useful as a vaccine for the prevention or amelioration of diseases or disorders g the presence of, or caused by aggregated HTT such as HD.
In one embodiment, it may be beneficial to use recombinant Fab (rFab) and single chain fragments (scFvs) of the antibody of the t invention, which might more readily penetrate a cell membrane. For example, Robert et (1]., Protein Eng. Des. Sel. (2008); 81741—0134, published online ahead, describe the use of chimeric recombinant Fab (rFab) and single chain fragments (scFvs) ofmonoclonal antibody WO-2 which recognizes an epitope in the N—terminal region of Abeta. The engineered fragments were able to (i) prevent amyloid fibrillization, (ii) disaggregate preformed Abeta1-42 fibrils and (iii) inhibit Abeta1-42 er-mediated neurotoxicity in vitro as efficiently as the whole IgG molecule. The perceived advantages of using small Fab and scFv engineered antibody s which lack the or function include more efficient passage across the blood-brain barrier and zing the risk of triggering inflammatory side reactions. Furthermore, besides scFv and -domain antibodies retain the binding specificity of full-length antibodies, they can be expressed as single genes and intracellularly in mammalian cells as intrabodies, with the potential for alteration of the folding, interactions, modifications, or lular localization of their targets; see for review, e.g., Miller and , Molecular Therapy 12 (2005), 394—401.
In a ent approach Muller et (11., Expert Opin. Biol. Ther. (2005), 237-241, describe a technology platform, so-called 'SuperAntibody Techno logy', which is said to enable antibodies to be shuttled into living cells without harming them. Such cell-penetrating dies open new diagnostic and therapeutic windows. The term 'TransMabs' has been coined for these antibodies.
In a further embodiment, co-administration or sequential administration of other antibodies useful for treating a disease, disorder, or symptoms related to the ence of mutated and/or ated HTT may be desirable. In one embodiment, the additional antibody is comprised in the pharmaceutical composition ofthe present ion. Examples of antibodies which can be used to treat a subject include, but are not limited to, antibodies targeting CD33, SGLT2, IL-6, and IL-1.
In a r embodiment, co-administration or sequential administration of other agents useful for ng a disease, disorder, or symptoms related to mutated and/or ated HTT, may be desirable. In one embodiment, the additional agent is comprised in the pharmaceutical composition of the present invention. Examples of agents which can be used to treat a subject include, but are not limited to: VMAT2 inhibitors targeting involuntary muscle movements such as neTM, anti-inflammatory agents such as diflusinal, corticosteroids, - dichloranilino) phenylacetic acid (diclofenac), iso-butyl-propanoic-phenolic acid (ibuprofen); diuretics, Epigallocatechin gallate, Melphalan hydrochloride, dexamethasone, omib, Bortezomib-Melphalan, Bortezomib-dexamethasone, Melphalan-dexamethasone, Bortezomib- Melphalan— dexamethasone; antidepressants, antipsychotic drugs, neuroleptics, antidementiva (e. g. the NMDA-rezeptor nist memantine), acetylcholinesterase inhibitors (e. g.
Donepezil, HCI, Rivastigmine, Galantamine), glutamat-antagonists and other nootropics blood pressure medication (e.g. Dihydralazin, dopa), cytostatics, glucocorticoides, angiotensin—converting-enzyme (ACE) inhibitors; nflammatory agents or any combination thereof.
A therapeutically effective dose or amount refers to that amount of the active ingredient sufficient to rate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, EDso (the dose therapeutically effective in 50% of the tion) and LDso (the dose lethal to 50% of the population). The dose ratio between eutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LDso/EDso.
From the foregoing, it is evident that the present invention encompasses any use of an HTT- binding molecule and/or fragments thereof comprising at least one CDR of the above described antibody, in particular for diagnosing and/or treatment of a disease or disorder related to mutated and/or aggregated HTT species and/or fragments thereof as mentioned above, such as HD and/or HTT amyloidosis. Preferably, said binding molecule is an antibody of the present invention or an immunoglobulin chain thereof. In addition, the present invention relates to anti- idiotypic antibodies of any one of the ned antibodies described hereinbefore. These are antibodies or other binding molecules which bind to the unique nic peptide sequence d on an antibody's variable region near the antigen-binding site and are useful, e.g., for the detection of anti-HTT antibodies in a sample obtained from a subject. In one embodiment thus, the present invention provides an dy as defined hereinabove and below or a HTT- binding le having substantially the same binding specificities of any one thereof, the polynucleotide, the vector or the cell as defined herein or a pharmaceutical or diagnostic composition comprising any one thereof for use in prophylactic treatment, therapeutic treatment and/or monitoring the progression or a se to treatment of a disease or disorder related to HTT, preferably n the disorder is associated with HTT dosis, such as Huntington's disease (HD).
In another ment the present invention relates to a diagnostic composition comprising any one of the above described HTT-binding molecules, antibodies, antigen-binding fragments, polynucleotides, vectors or cells of the invention and optionally suitable means for detection such as reagents conventionally used in immuno- or nucleic acid-based diagnostic methods.
The antibodies of the invention are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of immunoassays which can utilize the dy of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich ometric assay), flow cytometry, and the Western blot assay. The antigens and antibodies ofthe invention can be bound to many different carriers and used to isolate cells cally bound thereto. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. There are many different labels and methods of labeling known to those ofordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, dal metals, cent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed hereinabove.
By a further embodiment, the HTT-binding molecules, in particular antibodies of the present invention may also be used in a method for the diagnosis ofa disease or disorder in an individual by obtaining a body fluid sample from the tested individual which may be a blood sample, a plasma sample, a serum sample, a lymph sample or any other body fluid sample, such as a saliva or a urine sample and ting the body fluid sample with an antibody of the t invention under ions enabling the formation of antibody-antigen xes. The level of such complexes is then determined by methods known in the art, a level significantly higher than that formed in a control sample indicating the disease or disorder in the tested individual.
In the same manner, the specific antigen bound by the antibodies of the invention may also be used. Thus, the present invention relates to an in vitro immunoassay comprising the g le, 6.g. , antibody or antigen-binding fragment f of the invention.
In a further embodiment of the present invention the HTT-binding molecules, in particular antibodies of the present invention may also be used in a method for the diagnosis of a disease or disorder in an individual by obtaining a biopsy from the tested individual.
In this context, the present invention also s to means specifically designed for this purpose.
For example, an dy-based array may be used, which is for example loaded with antibodies or equivalent antigen-binding molecules of the present invention which specifically recognize HTT. Design of microarray immunoassays is summarized in Kusnezow et £21., Mol. Cell Proteomics 5 , 1681-1696. Accordingly, the present invention also s to microarrays loaded with HTT—binding molecules identified in accordance with the present invention.
In one embodiment, the present invention relates to a method ofdiagnosing a disease or disorder related to mutated and/or aggregated HTT species and/or fragments thereof in a subject, the method comprising determining the presence of HTT and/or mutated and/or aggregated HTT in a sample from the t to be diagnosed with at least one antibody ofthe present invention, a HTT-binding fragment thereof or an HTT-binding molecule having substantially the same binding specificities of any one f, wherein the presence of pathologically d and/or aggregated HTT is tive for HD and/or HTT amyloidosis and an se of the level of the pathologically mutated and/or aggregated HTT in comparison to the level of the physiological HTT is indicative for ssion ofHD and/or HTT amyloidosis in said subject.
The subject to be diagnosed may be omatic or preclinical for the disease. Preferably, the l subject has a disease associated with mutated and/or aggregated HTT, e.g. Huntington's disease (HD), wherein a similarity between the level of pathologically mutated and/or aggregated HTT and the reference rd indicates that the subject to be diagnosed has a HTT amyloidosis or is at risk to develop a HTT amyloidosis. Alternatively, or in addition as a second control the control subject does not have a HTT amyloidosis, wherein a difference between the level of logical HTT and/or of mutated and/or aggregated HTT and the reference standard indicates that the subject to be diagnosed has a HTT amyloidosis or is at risk to develop a HTT amyloidosis. Preferably, the subject to be diagnosed and the control subject(s) are age- matched. The sample to be analyzed may be any body fluid ted to contain pathologically mutated and/or aggregated HTT, for e a blood, blood plasma, blood serum, urine, peritoneal fluid, saliva or cerebral spinal fluid (CSF). In another aspect ofthe present invention, the dies of the present invention can be used in detection of soluble and aggregated HTT utilizing e.g. a TR—FRET based duplex immunoassay as described in Baldo et (1]., Chem. Biol. 19(2) (2012), 264-275 which disclosure content, in particular the experimental procedures at pages 273—274, are incorporated herein.
Furthermore, it has been described in e.g. Ren et al., Nature Cell Biol. 11 (2) (2009), 219-225 that ian cells can internalize ar polyglutamine peptide aggregates in culture gaining access to the cytosolic compartment and become co-sequestered in omes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. These internalized fibrillar aggregates were able to selectively recruit soluble cytoplasmic proteins and to confer a heritable phenotype upon cells expressing the homologous amyloidogenic protein from a chromosomal locus. Therefore, in one embodiment of the present invention the anti-HTT antibody can reduce extracellular spreading or transneuronal propagation of "toxic" HTT species, as shown by Pecho-Vriesling et al. Nat. Neurosci. (2014) doi:10.1038/nn.3761 for huntingtin or other ns involved in neurodegeneration such as u-synuclein; see e.g. Guo et al., Nat Med. 20(2) , 130-138.
The level iological HTT and/or of pathologically mutated and/or aggregated HTT may be assessed by any suitable method known in the art comprising, e.g, analyzing HTT by one or more techniques chosen from Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimrnunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption/ionization—time of flight—MS (MALDl—TOF), surface—enhanced laser tion ionization-time of flight (SELDI-TOF), high mance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), and laser densitometry. Preferably, said in viva imaging of HTT comprises scintigraphy, positron emission tomography (PET), single photon on aphy (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).
In one embodiment thus, an antibody of the present invention or a HTT-binding le having substantially the same binding specificities of any one thereof, the polynucleotide, the vector or the cell as defined hereinabove or a pharmaceutical or diagnostic composition sing any one thereof is provided for use in prophylactic treatment, eutic treatment, and/or monitoring the progression or a response to treatment of a disease or disorder related to HTT. In general thus, the t invention also relates to a method of diagnosing or monitoring the progression of a disease or er related to HTT (such as HTT amyloidosis) in a subject, the method comprising determining the presence of HTT in a sample from the subject to be diagnosed with at least one antibody ofthe present invention or a HTT-binding molecule having substantially the same g icities ofany one thereof, wherein the presence ofmutated, ded and/or aggregated HTT species or fragments thereof is indicative for the disease or disorder. In one embodiment said method of diagnosing or monitoring the progression of HTT amyloidosis in a subject is provided, the method comprising determining the presence of mutated and/or aggregated HTT and/or fragments thereof in a sample from the subject to be diagnosed with at least one antibody ofthe t invention or a HTT-binding le having substantially the same binding specificities of any one thereof, wherein the presence ofmutated and/or aggregated HTT and/or nt thereof is indicative of presymptomatic, prodromal or clinical HTT amyloidosis an increase of the level ofHTT aggregates in comparison to the level ofthe physiological HTT or in ison to a reference sample derived from a healthy control subject or a control sample from the same subject is indicative for progression of presymptomatic, prodromal or established HTT dosis. It would be appreciated by any person skilled in the art that in one embodiment said method is used as well for the diagnosing or monitoring the progression of any other disease or disorder from the group of disorders related to HTT as defined hereinabove.
As ted above, the antibodies of the present invention, fragments thereof and molecules of the same binding specificity as the antibodies and fragments thereof may be used not only in vitra but in viva as well, wherein besides diagnostic, therapeutic applications as well may be pursued. In one embodiment thus, the present invention also relates to a HTT binding molecule comprising at least one CDR of an dy of the present invention for the preparation of a composition for in viva detection of or targeting a therapeutic and/or diagnostic agent to HTT in the human or animal body. Potential therapeutic and/or diagnostic agents may be chosen from the austive enumerations of the therapeutic agents useful in treatment HTT amyloidosis and potential labels as indicated hereinbefore. In respect of the in viva g, in one preferred embodiment the t ion es said HTT binding le comprising at least one CDR of an antibody of the present invention, wherein said in viva imaging comprises scintigraphy, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared MIR) optical imaging or magnetic resonance imaging (MRI). In a further embodiment the present invention also provides said HTT-binding molecule comprising at least one CDR of an antibody of the present invention, or said molecule for the preparation of a composition for the above specified in viva imaging methods, for the use in the method of diagnosing or monitoring the progression of a disease or disorder related to HTT in a subject, as defined hereinabove.
VII. Peptides with aggregation specific HTT epitopes In a r aspect the present invention relates to peptides having an epitope of a polyP-rich region of HTT specifically ized by any antibody of the present invention. Preferably, such e comprises or consists of an amino acid sequence as indicated in SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156, 139, 151, 154, 158, 161, 157, 159, 160 as the unique linear e ized by the antibody or a modified sequence thereof in which one or more amino acids are substituted, d and/or added, wherein the peptide is recognized by any antibody of the present invention, preferably by antibody NI-302.74C1 1, NI-302.15F9, NI-302.39G12, NI-302.11A4, .22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI- 302.71F6, NI-302.33C11, NI-302.44D7, NI-302.7A8, NI-302.3D8, NI-302.46C9, NI- 302.11H6, NI-302.18A1, NI-302.52C9, and/or NI-302.8F1.
In an additional aspect the present invention relates to peptides having an epitope of the — region of HTT specifically recognized by any antibody of the present invention. Preferably, such peptide ses or consists of an amino acid sequence as indicated in SEQ ID Nos. 140, 141, 142, 143, 200 as the unique linear epitope recognized by the antibody or a modified sequence thereof in which one or more amino acids are substituted, deleted and/or added, wherein the peptide is recognized by any antibody of the t invention, preferably by antibody NI-302.63F3, NI-302.31Fl 1, NI-302.2A2, NI302.15D3 and/or NI-302.64E5.
Furthermore, in one embodiment the present invention relates to peptides having an e of the C-terminal region of HTT specifically recognized by any antibody of the t invention.
Preferably, such peptide ses or consists of an amino acid sequence as ted in SEQ ID NO: 145 or SEQ ID NO: 202 as the unique linear epitope recognized by the antibody or a modified sequence f in which one or more amino acids are substituted, deleted and/or added, wherein the peptide is recognized by any antibody of the present invention, preferably by antibody NI-302.35C1 or NI-302.72F10.
In an additional aspect the present invention s to peptides having an epitope of the N- terminal—region of HTT specifically recognized by any antibody of the present invention.
Preferably, such peptide comprises or ts of an amino acid ce as indicated in SEQ ID NOs: 144 as the unique linear epitope recognized by the antibody or a modified sequence thereof in which one or more amino acids are substituted, deleted and/or added, wherein the peptide is recognized by any antibody of the present invention, preferably by antibody NI- 302.15E8.
Furthermore, in one embodiment the present invention relates to peptides having an epitope of the Q/P-rich—region of HTT cally recognized by any antibody of the present invention.
Preferably, such peptide comprises or consists of an amino acid sequence as indicated in SEQ ID NO: 201 as the unique linear epitope ized by the antibody or a modified sequence thereof in which one or more amino acids are substituted, deleted and/or added, wherein the peptide is recognized by any antibody of the present invention, preferably by dy NI- 302.7D8.
In addition, in one embodiment the present invention relates to peptides having an e of HTT specifically recognized by any antibody ofthe present invention, preferably by antibody NI-302.6N9, NI-302.12H2, NI-302.8Ml and/or NI—302.4A6 in which one or more amino acids are substituted, deleted and/or added, wherein the e is recognized by any antibody of the present ion.
In one embodiment of this invention such a e may be used for diagnosing or monitoring a disease or disorder related to mutated, misfolded and/or aggregated HTT species and/or fragments thereof in a subject, such as HD and/or HTT amyloidosis comprising a step of determining the presence of an dy that binds to a peptide in a biological sample of said subject, and being used for diagnosis of such a e in said subject by measuring the levels of antibodies which recognize the above described peptide of the present invention and comparing the ements to the levels which are found in y subjects of comparable age and gender. Thus in one embodiment the present invention relates to a method for diagnosing HTT amyloidosis indicative of presymptomatic or clinical HD in a subject, comprising a step of determining the presence of an antibody that binds to a peptide as defined above in a biological sample of said subject. According to this method, an elevated level of measured antibodies specific for said e of the present invention is indicative for diagnosing in said subject presymptomatic or clinical HD or for diagnosing in said subject any other disease or disorder from the group of ers related to HTT as defined hereinabove.
The peptide of the t invention may be ated in an array, a kit and composition, respectively, as described hereinbefore. In this context, the present invention also relates to a kit usefiil in the diagnosis or monitoring the progression ofHD and/or HTT amyloidosis, said kit comprising at least one antibody ofthe present invention or a HTT-binding molecule having substantially the same binding specificities of any one thereof, the polynucleotide, the vector or the cell and/or the e as respectively defined hereinbefore, optionally with reagents and/or ctions for use.
The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of mistry and Molecular Biology, Oxford University Press, 1997, d 2000 and ted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application including the background section and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no ion that any document cited is indeed prior art as to the present ion.
A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLES Example 1: Isolation and fication of anti-HTT antibodies Human-derived antibodies targeting HTT and/or mutated and/or aggregated HTT species and/or fragments f were identified utilizing the method described in the international application the disclosure content of which is incorporated herein by reference, with modifications. In ular, wild-type and mutant HTT proteins obtained by recombinant expression were used in both native and d-aggregated conformations for the identification of HTT-targeting antibodies. The mutated-aggregated conformations were produced in vitro, using a procedure similar to the one described in Example 3. e 2: Determination of antibody sequence The amino acid sequences of the variable regions of the above identified TT antibodies were determined on the basis of their mRNA sequences, see Fig. 1. In brief, living B cells of selected non-immortalized memory B cell cultures were harvested. Subsequently, the mRNAs from cells producing selected anti-HTT antibodies were extracted and converted in cDNA, and the sequences encoding the antibody's variable regions were amplified by PCR, cloned into plasmid s and sequenced.
In brief, a combination of primers representing all sequence families of the human immunoglobulin germline repertoire was used for the amplifications of leader peptides, V- segments and J-segments. The first round of amplification was performed using leader peptide- specific primers in 5'-end and constant region-specific primers in 3'-end (Smith et al., Nat Protoc. 4 (2009), 372-384). For heavy chains and kappa light chains, the second round of amplification was performed using V-segment-specific primers at the 5'—end and J-segment- specific primers at the 3'-end. For lambda light chains, the second round amplification was performed using V—segment-specific primers at the 5'-end and a C-region—specific primer at the 3'-end (Marks et al., Mol. Biol. 222 , 581-597; de Haard et al., J. Biol. Chem. 26 (1999), 18218-18230). fication of the antibody clone with the desired specificity was performed by re-screening on ELISA upon inant expression of complete dies. Recombinant expression of te human IgG1 antibodies was ed upon insertion of the variable heavy and light chain sequences "in the correct reading frame" into expression vectors that complement the variable region sequence with a sequence encoding a leader peptide at the 5'-end and at the 3'- end with a sequence encoding the appropriate constant domain(s). To that end the primers contained ction sites designed to facilitate cloning of the variable heavy and light chain sequences into antibody expression vectors. Heavy chain immunoglobulins were expressed by inserting the immunoglobulin heavy chain RT-PCR product in frame into a heavy chain expression vector bearing a signal peptide and the constant domains of human or mouse immunoglobulin gamma 1. Kappa light chain immunoglobulins were expressed by inserting the kappa light chain RT-PCR—product in frame into a light chain expression vector providing a signal peptide and the constant domain ofhuman kappa light chain immunoglobulin. Lambda light chain immunoglobulins were expressed by inserting the lambda light chain RT-PCR- product in frame into a lambda light chain expression vector providing a signal peptide and the constant domain of human or mouse lambda light chain immunoglobulin.
Functional recombinant monoclonal antibodies were obtained upon co-transfection into HEK 293 or CHO cells (or any other appropriate ent cell line of human or mouse ) of an Ig-heavy-chain expression vector and a kappa or lambda ht-chain expression .
Recombinant human monoclonal antibody was subsequently purified from the conditioned medium using a standard Protein A column purification. Recombinant human monoclonal antibody can produced in unlimited quantities using either transiently or stably transfected cells.
Cell lines producing recombinant human monoclonal antibody can be established either by using the Ig—expression vectors directly or by re-cloning of Ig-Variable regions into different expression vectors. Derivatives such as F(ab), F(ab)2 and scFv can also be generated from these Ig-variable regions. The ork and complementarity determining regions were ined by comparison with reference antibody sequences available in databases such as Abysis (https://www.bioinf.org.uk/abysis/) and https://www.imgt.org/, and annotated using the Kabat numbering scheme (https://www.bioinf.org.uk/abs/).
Example 3: Expression of HTT exon 1 proteins Methods Recombinant huntingtin exonl proteins tExon1Q21 (GST—HD21), tExon1Q35 (GSTHD35), and GST-HttExon1Q49 (GST-HD49) sion and purification pGEX—6P-1 expression vector (GE Healthcare) encoding Exonl of human gtin with polyQ length of 21, 35 or 49 CAG repeats, tively (compare Fig. 2A) fiased with a PreScission cleavage site to an N-terminal Glutathione S-transferases (GST)-tag were expressed in E. coli strain BL21. Overnight bacterial cultures (37°C, 220rpm) were diluted 1:25 and expression was induced at an Absorption 600 of 6 for 4 hrs by on of 1 mM 1PTG (Sigma 11284) and further incubation at 36°C, 220 rpm. Cultures were grown in LB medium containing 100 ug/ml ampicillin at 37°C, for overnight cultures in addition with 1% glucose. Recombinant GST—HttExonl proteins were purified by binding to glutathione agarose (Sigma G4510).
Briefly, the bacteria pellet was ended in 20-40ml of cold buffer 1 (50mM NaH2PO4, mM Tris, 150 mM NaCl, 1 mM EDTA pH8, 5mg/ml final lysozyme, protease inhibitor complete (Roche)) were incubated for 60 min on ice, ultrasonicated, Triton-X100 added (0.1% final) and centrifuged for 90 min at 14’000g after incubation on ice for 5 min. Glutathione agarose was added to the supernatant, incubated for 2 hrs at 4°C, spun down for 10 min at 1000g and washed 2x with cold PBS after removal ofthe supernatant. Elution was performed for 5 min in 1 ml buffer 1 with 10mM reduced glutathione pH 9. This step was repeated 5 to 15 times until no further protein was eluted. The pooled supematants were dialyzed against buffer (50mM tris pH7.4, 150 mM NaCl, 1 mM EDTA, 1% glycerol) over night (10 kD MWCO, Pierce) and aliquots were stored at —80°C.
SDS-PAGE analysis Purified recombinant GST-HttExl proteins were resolved by nt GE (NuPAGE Bis-Tris 4-12%; Invitrogen, Basel, Switzerland) followed by staining with Coomassie brilliant blue or electroblotting on nitrocellulose membranes. Blots were incubated with primary antibodies Mab 5492 (Chemicon N—terminal aa1-82 epitope, P-rich domain) or NI-302.37C12 ed by a goat antimouse IgG secondary antibody conjugated with HRP or donkey anti- human IgG secondary antibody conjugated with HRP. Blots were developed using ECL and ImageQuant LAS 4000 detection (GE Healthcare, Switzerland).
As shown in Fig. 2B the different recombinant GST-HttExl proteins were successfully expressed and purified as demonstrated by Coomassie ng after SDS-PAGE.
Example 4: Characterization of aggregation state by dot blot and filter retardation To characterize HD21, HD35 and HD49 protein aggregation kinetics filter retardation and dot- blot analyses were performed.
Therefore, at the beginning an aggregation reaction was performed as follows: Recombinant GST-HttExonl ns can be expressed and purified as a fiasion protein. As soon as the GST tag is cleaved off from the fusion protein by the ssion Protease (PP) the aggregation reaction of the huntingtin Exonl protein starts immediately. Before the start of ons the GST-HttExonl ns were centrifuged at 100’000g for 30 minutes. The cleared protein on were diluted to 2uM protein concentration in cold aggregation buffer (0.05 M CL pH 7, 0.15 M NaCl, 1 mM EDTA) and 1 mM DTT and PreScission Protease (GE Healthcare) were added. The reaction was incubated at room temperature with rotating at 300rpm and the aggregation reactions were stopped by snap freezing at -80°C after the indicated time intervals. Aliquots of HD21, HD35 and HD49 aggregation reactions were subsequently removed after 1, 3, 5, 7 and 24 hrs of incubation time, respectively, snap frozen on dry ice and stored at -80°C.
For the dot blot analysis samples were thawed on ice, diluted and transferred onto a nitrocellulose transfer membrane with a filter device applying vacuum in the chamber below the membrane. To that end, the membrane was equilibrated with PBS, mounted in the chamber and washed with 100ul PBS per well. The samples were loaded and completely sucked h the membrane ed by 3 washes with PBS. The device was bled and the membrane was briefly air-dried for 15 min at room temperature, blocked for 1 hour at room temperature with blocking buffer (3% BSA, 0.1% Tween 20 in PBS buffer) and incubated with polyclonal HD-l antibody (1 :10’000, kind gift of Prof. E. Wanker,MDC, Berlin). After washing, the membrane was ted for 1 h at RT with an anti-rabbit IgG antibody coupled to HRP and blots were developed using ECL and ImageQuant LAS 4000 detection (GE Healthcare, Switzerland).
As evident from the dot blot shown in Fig. 2C, left side, polyclonal HD—l antibody ed HD21, HD35 and HD49 proteins irrespective of their aggregation state.
For filter retardation assays samples were thawed on ice, diluted in denaturation buffer (4% SDS, 100 mM DTT) and transferred through a cellulose acetate membrane with a pore size of 0.2um using a vacuum chamber: To that end, the membrane was equilibrated in 0.1% SDS in PBS, mounted on the vacuum chamber and the wells were washed with 0.1% SDS. The samples were added, filtered through the membrane by vacuum and washed 3 times with 0.1% SDS.
The ne was then removed from the chamber, briefly air-dried, blocked for 1h at RT in blocking buffer (5% milk, 0.1% Tween 20 in PBS buffer), incubated with polyclonal HD-l antibody (1:5’000, Scherzinger et al., Cell 90 (1997), 549—558) and processed further as described above.
In the filter retardation assay, the first aggregates retained by the membrane were detected for HD35 after 24 hours of incubation. HD49 proteins with an ed polyQ tract form insoluble aggregated as early as 3 hrs after cleavage of the GST tag, see Fig. 2C right side (FRA).
Example 5: Characterization of Huntingtin Exonl aggregates To verify and characterize HD35 and HD49 Exonl ate formation electron microscopy (EM) was performed. In brief, HD49 aggregation reactions after 1, 3 and 24 hrs, respectively or s from HD35 afier 24 hrs were analyzed by electron microscopy. Samples were ed onto glow—discharged carboncoated copper grids. Excess sample was removed by blotting on filter paper. Grids were stained with 2% (w/V) uranyl acetate for l min and excess uranyl acetate was washed with distilled deionized water. Grids were air-dried and imaged using a Philips CMIOO transmission electron microscope with an acceleration voltage of 100 EM analysis of the HD35 aggregation reaction ed larger aggregates visible by EM resembling protofibrillar structures after 24 hrs of incubation (Fig. 2D [E]). HD49 displayed a more rapid aggregation kinetics with fibrils being able already after 1 hour of incubation (Fig. 2D [F]) and increasing in size and number with aggregation time (Fig. 2D [C, D, G, H]).
These observations were consistent with the results obtained in the filter retardation assays where ates larger than 0.2um are retained on the cellulose acetate membrane and confirm the successfiil preparation of huntintingtin exon 1 ates; see also Example 4. e 6: Binding affinity of anti-polyP domain NI—302.33C11 antibody utilizing direct ELISA and EC50 To determine the half maximal effective concentration (EC50) of recombinant human-derived HTT antibody NI-302.33C11 to soluble and aggregated huntingtin Exonl proteins with 21 or 49 polyQ repeats direct ELISA was performed. In brief, 96 well lates (Corning) were coated with either 21, GST-HD49 or aggregated HD21 or HD49 at a tration of 5 ug/ml in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.42). Nonspecific binding sites were blocked for 1 h at RT with PBS/0.1% -20 containing 2% BSA (Sigma- Aldrich, Buchs, Switzerland). Primary antibodies were diluted to the indicated concentrations and incubated 1 h at RT. Binding was determined using either a donkey anti-human IgG Fcy- specific antibody conjugated with HRP or a goat anti-mouse IgG (H+L)-specific antibody conjugated with HRP, followed by measurement of HRP activity in a standard metric assay. Subsequently, EC50 values were estimated by a near regression using GraphPad Prism software (San Diego, USA).
The EC50 ofhuman-derived HTT antibody NI-302.33C1 1 for aggregated and soluble HTT exon 1 ns with 21 or 49 poly Q repeats was determined by direct ELISA with coating of the different preparations at 5 ug/ml concentration. As shown in Fig. 3A and B antibody NI-302.33C11 bound with similar high affinity to all four species including the pathologically aggregated HTT Exonl HD49 with an EC50 of approximately 100 pM.
Example 7: Binding selectivity of anti-HTT antibodies utilizing dot blot and filter retardation assay To characterize recombinant human-derived HTT antibody .33C11 to soluble and aggregated huntingtin Exonl proteins with 21, 35 or 49 polyQ repeats filter retardation assay and dot-blot were performed. For this reason, aliquots of HD21, HD35 and HD49 aggregation ons as described in Example 4 were removed afier 1, 3, 5, 7 and 24 hrs of incubation time, snap frozen on dry ice and stored at -80°C and a dot blot was med as bed in Example 4. Filter retardation assay was also performed as described in e 4, with the exception that the membrane was incubated with NI-302.33Cll (l .
It could be shown that on the dot blot (Fig. 3C, left side), antibody NI-302.33C11 preferentially detects proteins of huntingtin with expanded polyQ tracts (HD49>>HD35>HD21).
Furthermore, the signal intensity increased with increasing incubation times of the ation reactions ofHD35 and HD49.
This is also true for the results shown in the filter retardation analysis (Fig. 3C, right side), which showed that NI-302.33C11 detects HD35 and HD49 aggregates that were retained on the 0.2 mm pore size membrane. These s based on spotted protein preparations suggested that antibody NI—302.33C11 has a preference for aggregated HTT conformations with pathogenic polyQ expansions.
Example 8: Binding city and selectivity of anti-HTT dies to unrelated aggregating protein targets utilizing direct ELISA To determine the binding specificity antibody NI-302.33C11 recombinant antibody g to the polyP-region of HTT and not to unrelated aggregating protein targets direct ELISA was performed on 96 well microplates (Corning) coated with different target proteins at a concentration of 1-10 ug/ml in coating buffer (15 mM N32CO3, 35 mM NaHCOg, pH 9.42).
Non-specific binding sites were blocked for 1 h at RT with PBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland). NI-302.33Cll antibody was diluted to the indicated concentrations and incubated l h at RT. Binding was determined using donkey anti- human IgG Fcy-specific antibody conjugated with HRP ed by measurement of HRP activity in a rd colorimetric assay. Signals for target protein were calculated in fold increase above median.
It could be shown that human-derived NI-302.33C11 binds cally to HTT, z'. e. aggregated HD49, with absent binding to the other unrelated protein targets including prominent amyloid- g proteins, see Fig. 16A.
Example 9: Assessment of the binding epitope of the HTT antibody NI—302.33C11 To map the huntingtin (HTT) epitope recognized by the NI-302.33Cl l human-derived antibody epitope mapping by peptide scanning analysis with synthetic es was performed.
In brief, scans of overlapping peptides were used for epitope mapping. The sequence of human HTT Exon 1 sequence was synthesized as a total of 16 linear lS-meric peptides with 10 aa overlap between individual peptides (JPT Peptide Technologies, Berlin, Germany) and spotted onto nitrocellulose membranes. The membrane was activated for 5 min in methanol and then washed at RT in TBS for 10 min. Non-specific g sites were blocked for 2 hours at room temperature with Roti®-Block (Carl Roth GmbH+Co. KG, Karlsruhe, y). Human NI- 302.33Cll antibody (1 ug/ml) was incubated for 3 hrs at RT in Roti®-Block. Binding of y antibody was ined using HRP conjugated donkey-anti human IgGy secondary dy. Blots were developed using ECL and ImageQuant LAS 4000 detection (GE Healthcare, Switzerland).
As shown in Fig. 4, prominent binding of NI-302.33Cll was observed to peptides number 7, 8, 9, l3 and 14 indicating that the epitope ized by this antibody is localized in the polyP repeat domain of huntingtin. The NI-302.33Cll binding epitope is therefore predicted to be localized within HTT amino acids 35-PPPPPPPP-42 (SEQ ID No.: 139) and amino acids 63- PPPPPPPPPPP-72 (SEQ ID No.: 162).
Example 10: Epitope mapping by direct ELISA binding to different Exonl peptides of the HTT antibody NI—302.33C11 To determine the half maximal effective concentration (ECso) of recombinant human-derived HTT antibody NI-302.33Cll to BSA-coupled peptide fragments ofthe huntingtin Exon 1 direct ELISA with upled Htt Exonl domain peptides was performed.
In brief, 96 well microplates (Corning) were coated with BSA-coupled synthetic peptides (Schafer-N, k) of the N—terminal amino acid l-l9 (MATLEKLMKAFESLKSFQQ, SEQ ID No.: 93), the P-rich domain sequence (PPQLPQPPPQAQPLLPQPQPP, SEQ ID No.: 94), the polyP repeat sequence (PPPPPPPPPPP, SEQ ID No.: 95) or the 14 C—terminal amino acids (PPGPAVAEEPLHRP, SEQ ID No.2 96) or with the full lengths GST-HD49 Exon 1 protein at Sug/ml in coating buffer (15 mM Na2C03, 35 mM NaHCO3, pH 9.42). Non-specific binding sites were blocked for 1 h at RT with 1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland). Primary antibodies were diluted to the indicated concentrations and incubated l h at RT. Binding was determined using donkey anti-human IgG Fcy-specific antibody conjugated with HRP, followed by measurement of HRP activity in a standard colorimetric assay and the ECso values were estimated by a non-linear regression using GraphPad Prism software (San Diego, USA).
As shown in Fig. 5 NI-302.33C11 bound with high y to the BSA-coupled polyP peptide as well as to fiJll-length GST-HD49 with an equivalent ECso of 30 pM. This confirms the epitope mapping to the polyP sequence as shown in Example 9.
Example 11: Assessment of the purity and integrity of recombinant human NI- 302.33C11 anti-polyP domain antibody To assess the purity and integrity ofrecombinant human NI-302.33C11 anti-polyP domain lead antibody human NI-302.33C11 anti-polyP domain antibody was expressed by transient ection of CHO-S cells and purified by protein A affinity purification on an Akta system.
After PD-10 column desalting the antibody was formulated in PBS. Subsequently SDS-PAGE is was performed, wherein two and 10 ug fied recombinant human .33C1 l anti—polyP domain antibody were resolved under reducing conditions by gradient SDS-PAGE (NuPAGE 4—12% Bis-Tris gel; Invitrogen) followed by sie staining (SimplyBlue ain, Invitrogen).
The SDS-PAGE analysis under ng conditions of the recombinant human NI-302 anti- polyP domain lead antibody revealed two major bands corresponding to the antibody heavy and light chains at the expected size as shown in Fig. 6, while no significant contaminations or proteolytic ation products were ed.
Example 12: Characterization of HTT antibody NI—302.33C11 in human HTT transgenic mice To assess the g of NI-302.33C11 dy to huntingtin pathology in human HTT transgenic mouse brain tissues immunohistochemistry was performed. The B6.Cg- Tg(HDexon1)61pr/J transgenic mouse line (Mangiarini et al., Cell 87 (1996), 493-506) is a well terized mouse model for Huntington's Disease (HD). Starting at around 9 weeks of age, this animal model develops a progressive pathology characterized by intranuclear inclusions of huntingtin reminiscent of human Huntington's disease (Naver et al., Neuroscience 122 (2003), 057). Hemibrains of these B6.Cg-Tg(HDexon1)61pr/J transgenic mice at a progressed stage of disease (270 days) were fixed in phosphate-buffered 4% paraformaldehyde solution, paraffin—embedded, and S-um sections were prepared. After formic acid and citrate buffer pretreatment, sections were incubated with 1, 5 or 50 nM human NI- 302.33C1l anti-HTT antibody followed by incubation with biotinylated —anti—human secondary antibody (Jackson Immunoresearch; 1:250). dy signal was amplified with the Vectastain ABC kit (Vector Laboratories) and detected with diaminobenzidine (Pierce).
As shown in Fig. 27 human-derived polyP domain antibody NI-302.33C11 revealed a very prominent staining of neuronal intranuclear inclusion pathology already at the lowest 1 nM concentration (Fig. 27 [E-H]) consistent with the high affinity binding to huntingtin ates as determined by ELISA and dot blot es. At a concentration of 5 nM or higher, the antibody stained in addition entire medium spiny s and produced a more generalized diffuse ng which was also detectable on nontransgenic brain sections. A certain degree of reactivity cannot be excluded as the polyp epitope targeted by NI-302.33C11 was present also in numerous unrelated proteins (Fig. 27 [F-H]). e 13: Characterization of g affinity and selectivity of anti-P-rich domain NI—302.63F3 antibody utilizing direct ELISA and ECso To determine the half maximal ive concentration (ECso) of recombinant human-derived HTT antibody .63F3 to soluble and aggregated HTT Exonl proteins with 21 or 49 polyQ repeats direct ELISA and EC50 determination was performed as described in Example 6, supra.
It could be shown that NI-302.63F3 binds with similar high affinity to all four species including the aggregated HTT Exonl HD49 with an EC50 of approximately 200 to 400 pM Fig. 7 A and B. Accordingly, the human-derived HTT anti-P-rich domain dy NI-302.63F3 targets an epitope exposed in aggregated as well as in an uncut, more linear structure of HTT Exonl protein with high-affinity in the subnanomolar range. onally, to characterize the binding of recombinant human-derived HTT antibody NI- 302.63F3 to soluble and aggregated HTT Exonl proteins with 21, 35 or 49 polyQ repeats using filter retardation assay and dot blot as described in Example 7, supra, with the small modification that the incubation was performed with 1 ug/ml ofNI-302.63F3 antibody.
On the dot blot, antibody NI-302.63F3 most ently detected the HD49 protein with an expanded polyQ tract (Fig. 7C, left side). In the filter retardation analysis NI-302.63F3 detected HD35 and HD49 ates that were retained on the 0.2 um pore size membrane (Fig. 7C, right side). These findings based on spotted protein preparations demonstrate that antibody NI- 302.63F3 recognizes aggregated HTT conformations with pathogenic polyQ expansions.
Furthermore, to determine the binding of NI-302.63F3 recombinant dy to unrelated aggregating protein targets, direct ELISA was performed as described in Example 8, supra. As shown in Fig. 16B human-derived NI-302.63F3 bound cally to HTT while a binding to unrelated proteins could not be shown.
Example 14: Assessment of the binding e of the HTT antibody NI—302.63F3 To map the huntingtin epitope recognized by the NI-302.63F3 human-derived antibody epitope mapping with synthetic peptides was performed as described above in Example 9.
As shown in Fig. 8, prominent binding of NI-302.63F3 was observed to peptides number 10 and 11 with a weak signal on peptide 8 and 9 indicating that the epitope recognized by this antibody is localized in the P-rich domain (between the polyP repeat regions) of HTT. The NI- 302.63F3 binding epitope was therefore predicted to be localized within HTT amino acid ce 43-(PPPQL)PQPPPQAQPL-57 (SEQ ID Nos.: 161 and 140).
Example 15: Epitope g by direct ELISA binding to different Exonl peptides of the HTT antibody NI—302.63F3 To ine the half maximal effective concentration (EC5 0) of recombinant human-derived HTT dy NI-302.63F3 to BSA—coupled peptide fragments of the gtin Exon 1 direct ELISA with BSA-coupled Htt Exonl domain peptides and ECso determination were performed as described in Example 10.
As shown in Fig. 9 .63F3 binds with high affinity to the BSA-coupled P-rich domain peptide as well as to fiill-length GST—HD49 with a similar ECso of200 to 300 pM. This confirms the epitope mapping to the P-rich domain ce as shown in Example 14.
Example 16: Assessment of the purity and ity of recombinant human NI-302.63F3 anti-P-rich domain antibody To assess the purity and integrity of recombinant human NI-302.63F3 roline-rich domain antibody SDS-PAGE analysis was performed as already described in Example 11, supra.
SDS—PAGE analysis under reducing ions of the recombinant human NI-302.63F3 anti- P-rich domain antibody ed two major bands ponding to the antibody heavy and light chains at the expected size. No significant contaminations or proteolytic degradation products were detected as shown in Fig. 10.
Example 17: Characterization of HTT antibody NI—302.63F3 in human HTT transgenic mice The ment of the binding of NI—302.63F3 -rich domain antibody to HTT pathology in human HTT transgenic mouse brain tissues was assessed as described in Example 12, supra with the difference that the incubation of the sections was performed with l or 50 nM of the anti-P-rich domain antibody.
As shown in Fig. 28 [E-F] the derived NI-302.63F3 -rich domain dy revealed a prominent and highly specific staining of neuronal intranuclear inclusion pathology at l and 50 nM concentration in the striatum and cortex ofR6/ 1 transgenic animals consistent with the high affinity binding to HTT aggregates as determined by ELISA and dot blot analysis.
However as shown in Fig. 28 [F] at a concentration of 50 nM the antibody NI-302.63F3 stained additionally weakly the entire nucleus of the medium spiny neurons.
Example 18: Characterization of binding affinity and selectivity of anti-C-terminal domain antibodies NI—302.35C1 and NI-302.72F10 utilizing direct ELISA and ECso To ine the half maximal effective concentration (ECso) of recombinant human-derived HTT antibodies NI—302.35Cl and NI-302.72F10 to soluble and aggregated HTT Exonl proteins with 21 or 49 polyQ repeats direct ELISA and ECSO determination was performed as described in Example 6, supra.
It could be shown that NI—302.35C1 binds with high affinity to all four species including the aggregated HTT Exonl HD49 with an ECso of approximately 2.7 nM; see Fig. 11 A and B.
Similarly, NI-302.72F10 binds to all four species albeit with a different affinity than NI- 302.35C1 gated HD21>>GST-HD21>>aggregated GST-HD49) (Fig. 31 C) which may be explained with the different epitopes recognized by both antibodies (Fig. 20).
Accordingly, the human-derived HTT anti—C-terminal domain antibodies NI-302.35C1 and N1- 302.72F10 target an epitope exposed in aggregated as well as in soluble forms ofHTT with low nanomolar affinity.
Additionally, to characterize the binding of recombinant human-derived HTT antibodies NI- 302.35C1 and NI—302.72F10 to soluble and aggregated HTT Exonl proteins with 21, 35 or 49 polyQ repeats filter retardation assay and dot blot as described in Example 7, supra, was performed.
On the dot blot, antibody NI-302.35C1 preferentially detected constructs ofHTT with expanded polyQ tracts (HD49>HD35>>HD21, Fig. 11C, left side). Furthermore, the signal ity increases with increasing incubation times of the aggregation reactions of HD35 and HD49.
Similarly, antibody NI-302.72F10 detected ucts of HTT with expanded polyQ tracts albeit with a different preference (HD35>>HD49>HD21) whereas the signal intensity increases with increasing incubation times of the aggregation reactions of HD35 only (Fig. 32 C).
In the filter retardation analysis NI—302.35C1 detected HD35 and HD49 aggregates that were retained on the 0.2 um pore size ne (Fig. 11 C, right side) s NI—302.72F10 detected HD35 aggregates only (Fig. 32 C, right side). These findings based on membrane bound n preparations suggested that antibodies NI-302.35C1 and NI-302.72F10 preferentially targets aggregated HTT conformations with pathogenic polyQ expansions. rmore, to ine the binding of NI-302.35Cl and .72F10 recombinant antibodies to ted aggregating protein targets, direct ELISA was performed as described in Example 8, supra. As shown in Fig. 16 C human—derived NI-302.35C1 as well as shown in Fig. 33 C human-derived NI-302.72F10 bound specifically to HTT while a binding to unrelated proteins could not be shown.
Example 19: Assessment of the binding epitope of the HTT antibody NI-302.35C1 To map the huntingtin epitope recognized by the NI—302.35Cl human-derived antibody epitope g with synthetic es was med as described above in Example 9.
Determination of NI-302.35Cl antibody binding epitope by scan of overlapping peptides did not result in specific signal. Therefore, processing this antibody in the way it was done for the other HTT NI-302 antibodies did not results in any specific signal on the individual es for unknown reasons. The epitope was successfully mapped to a inal peptide by coupling it to BSA, see also Example 20.
Example 20: Epitope mapping by direct ELISA binding to different Exonl peptides of the C-terminal domain HTT antibody NI—302.35C1 To determine the half maximal effective concentration (ECso) of recombinant human-derived HTT antibody NI-302.35Cl to BSA-coupled peptide nts of the HTT Exon 1 direct ELISA with upled Htt Exonl domain peptides and ECso determination were performed as described in Example 10.
As shown in Fig. 12 NI-302.35Cl binds with high affinity to the BSA—coupled C-terminal e as well as to full-length 49 with an ECso value of approximately 0.7 nM and 3.2 nM, respectively. This s the epitope to the C-terminal region ofHTT Exonl sequence (71- PPGPAVAEEPLHRP-SS, SEQ ID No: 96). If the same inal peptide was coated directly to the plate only weak binding was observed (ECso >100nM, data not shown) suggesting that the presentation of the peptide coupled to BSA increases the g to the epitope and might be an explanation why the epitope mapping by e scanning analysis as shown in Example 20 did not work.
Example 21: Assessment of the purity and integrity of recombinant human NI—302.35C1 anti-P-rich domain antibody To assess the purity and integrity ofrecombinant human NI—302.35Cl anti—C-terminal domain antibody SDS-PAGE analysis was performed as already described in Example 11, supra.
SDS-PAGE analysis under reducing conditions of the recombinant human NI-302.35Cl anti- C-terminal domain antibody revealed two major bands corresponding to the antibody heavy and light chains at the ed size, while no significant contaminations or proteolytic degradation products were ed (Fig. 13). e 22: Characterization of HTT antibody NI—302.35C1 in human HTT transgenic mice The assessment of the g of NI-302.35C1 anti-C-terminal domain antibody to HTT pathology in human HTT transgenic mouse brain tissues was assessed as described in Example 12, supra with the difference that the incubation of the sections was performed with 5 or 50 nM ofthe anti-C-terminal domain antibody.
As shown in Fig. 29 [E-F] the human-derived NI-302.35C1 anti-C-terminal domain antibody revealed a prominent staining of neuronal intranuclear inclusion pathology at 5 and 50 nM concentration in striatum of R6/1 transgenic animals tent with the high affinity binding to HTT ates as determined by ELISA and ot analyses.
Example 23: Assessment of the effects of human-derived antibodies targeting HTT 0n spine density in hippocampal slice cultures Antibody Expression and Purification Human-derived antibodies targeting distinct domains in HTT exon 1 were expressed by transient transfection ofCHO-S cells and purified by protein A affinity purification on the Akta system. After PD-10 column desalting the antibodies were formulated in PBS. Endotoxin levels were confirmed to be <10 EU/ml. ampal slice culture Organotypic hippocampal slice cultures were prepared and cultured according to Stoppini et al., J Neurosci Methods. (1991) 37(2):173—82. In short, 6- to 8-d-old B6CBA- Tg(HDexon1)62pr/1J transgenic and nontransgenic litterrnates were decapitated, brains were d, and both hippocampi were isolated and cut into 400-um thick slices. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Slices were cultured on Millicell e plate inserts (0.4 um, Millipore) in six-well plates containing 1 ml of culture medium (46% minimum essential medium Eagle with HEPES modification, 25% basal medium with Earle's modification, 25% heat-inactivated horse serum, 2 mM glutamine, 0.6% glucose, pH 7.2). Culture plates were kept at 37°C in a humidified atmosphere containing 5% C02. Slices were kept in culture for 7 d before the experiments. Culture medium was exchanged every second or third day. On day 7 antibodies were added at a concentration of l. On day 10 in vitro slice cultures were ed with Sindbis virus using a droplet method (Shahani et al., J ci. 31 (2006), 6103— 6114). For spine analysis, cultures were fixed at day 4 post infection (14 days in Vitro). Slices were left attached to the culture plate membrane to preserve hippocampal structure and rinsed with PBS. Slices were then fixed with 4% paraformaldehyde in PBS containing 4% sucrose for 2 h at 4°C. For each dendrite a picture was taken and spines were analyzed over a length of 30- 45 um. Eight to 13 slices per group from a total of 12 enic animals were quantified for each antibody treatment. Data represent the mean i SEM. *p<0.05 (MWU), # p=0.05.
The quantification of tic spine density in hipppocampal slice cultures of Tg(HDexon1)62pr/1J transgenic mice (Fig. 17 B, D) revealed a significant reduction by 53% compared to non-transgenic littermates (Fig. 17 A, C, E) using hippocampal slices of postnatal day 6 animals after 14 days in Vitro with the experimental design described above. Upon on of human-derived NI-302 antibodies at concentration of 10 ug/ml for seven days a significant ation of spine density loss was observed for antibodies NI-302.31F11 (Fig. 17 F, p < 0.05, t-test) and NI-302.63F3 (p=0.05, t-test), whereas antibody NI—302.33C11 and NI- 302.35C1 did not show a clear effect under the conditions tested.
The significant reduction of spine density compared to non-transgenic littermate in displayed in hippocampal slice cultures of Tg(HDexon1)62pr/1J enic mice led to the suggestion that this is a suitable model for in Vitro testing of HTT ate antibodies for their activity towards interference with HTT toxicity. In this model, antibodies .63F3 and NI- 302.11F11 that both targeted the P-rich domain within HTT exon 1 were able to improved spine density compared to an isotype control antibody. This suggests that these antibodies can attenuate the toxic effects on spine density driven by sion of pathological poly-Q- expanded HTT. e 24: Penetration of NI-302 antibodies in the brain of R6/1 animal model To test the penetration of the anti-HTT antibodies of the present invention a transgenic mice model was utilized. In particular, Tg(HDexon1)61pr/J transgenic mice harbor a 1.9 kb transgene which was isolated from a phage genomic clone derived from an Huntington's disease (HD) t and contained the 5' end of the human huntingtin (HTT) gene. It was composed of approximately 1 kb of 5' UTR sequences, exon 1 (carrying expanded CAG repeats of ~130 units) and the first 262 bp of intron 1. This construct was microinjected into single cell 7BL/6 embryos. Male founder R6 was bred to CBAxC57BL/6 females, producing several founder lines. Mice from founder line R6/1 have the transgene integrated as a single intact copy which is ubiquitously expressed. Transgenic mice on a mixed CBAxC57BL/6 genetic background were backcrossed to C57BL/6J for more than 12 to generate the congenic strain B6 .Cg—Tg(HDexon 1 )6 l pr/J .
R6/1 transgenic mice t a ssive neurological ype that mimics many of the features of HD, including choreiform—like movements, involuntary stereotypic movements, tremor, and epileptic seizures, as well as nonmovement disorder components, including unusual vocalization. They urinate frequently and exhibit loss of body weight and muscle bulk through the course of the disease. Neurologically they develop al uclear inclusions (N11) which contain both the HTT and ubiquitin proteins. These N11 have also been identified in human HD patients. The age of onset ofHD symptoms is reported to occur between 15 and 21 weeks for this 6/1 line.
The study animals displayed the following properties and were identified by classical ear marking: Strain: Hemizygous B6.Cg-Tg(HDexon1)61pr/J (Mangiarini et (11., Cell, 87 (1996), 493-506) Source: Jackson Laboratory, Maine, USA Sex: Males and s Age start: 230 to 260 days s: NI-302.31F11 Total: 3 males NI-302.35C1 Total: 3 males Vehicle Total: 3 males For the spinal cord homogenization Tg(HDexon1)61pr/J transgenic mice were deeply anesthetized and transcardially ed with cold phosphate-buffered saline through the left ventricle by mean of a altic pump. The brain was dissected out and snap frozen on dry ice.
The tissue samples were homogenized in 1:10 w/v DEA-Buffer (SOmM NaCl, 0.2% DEA, protease inhibitor complete, Roche Diagnostics) with a hand-sonicator (Sartorius, Labsonic M).
Samples were centrifuged for 30 minutes at 100’000 x g at 4°C and aliquots of the supernatant were stored at -80°C before analysis.
In the following human IgG drug level sandwich ELISA the human NI-302.35C1, NI- F11 antibody plasma levels were determined using the corresponding recombinant dy ofknown tration as standard. 96 well microplates (Corning) were coated with donkey anti human IgG (709 149, Jackson Irnrnunoresearch) at lug/m1 in 50mM carbonate g buffer pH 9.6. Non-specific binding sites were blocked for 1 hr at RT with PBS/0. 1% Tween®-20 containing 2% BSA , Buchs, Switzerland). Plasma samples were d 1:20’000 and 1:100’000, brain homogenates were diluted 1:5 and 1:50 and both were incubated 1 hr at RT together with the standard dilution curves. Binding was determined using the detection antibody anti human HRP (709—036-098, Jackson Immunoresearch), followed by measurement of HRP activity in a standard colorimetric assay. Concentrations of plasma and spinal cord samples were calculated based on the individual standard curve. Values shown in table 6-1 are average values of 2 independent ELISA experiments.
Plasma and brain samples were obtained 2 days after a single intraperitoneal injection of 50 mg/kg of antibodies NI-302.31F11, NI-302.35C1 in R6/1 transgenic mice. Antibody levels in plasma and brain homogenates were ined by human sandwich IgG ELISA (Fig. 18 A and B). The ratio of brain versus plasma drug levels was ined at 0.13fl:0.02% and .06% for human-derived dies NI-302.31F11 and NI-302.35C1, tively.
These results suggest a 48h brain penetration of the tested NI-302 antibodies in the expected range in HTT enic mice.
Example 25: Characterization of binding affinity and specificity of further antibodies identified in accordance with the present invention To determine the half maximal effective concentration (ECso) of fithher identified recombinant human-derived HTT antibodies to soluble and aggregated HTT Exonl proteins with 21 or 49 polyQ repeats direct ELISA with g of the different preparations at 5 ug/ml concentration and ECso determination was performed as described in Example 6, supra.
The determined EC50 for the different HTT species are shown in Fig. 19 as well as Fig. 31 (A, D-F) and summarized in Fig. 20. Most human-derived antibodies bound with high affinity at subnanomolar or low nanomolar ECso. Some candidates such as NI-302.37C12, NI-302.55D8, NI-302.11A4, NI-302.22H9 or NI-302.64E5 seemed to have preferred binding to uncut GST- HTT protein, other antibodies such as NI—302.74C1 1, NI—302.71F6, NI-302.4A6, NI-302.12H2 or NI8M1 showed high affinity binding with similar ECso-values to all HTT preparation in the ELISA assay. NI-302.15F9 showed about a 5-fold ence of HD49 vs. HD21 with ECso values in the range of 5 to 35nM. Antibody 33Cll served as control in this experiment (Fig. 32 G).
Therefore, a panel of high affinity recombinant HTT specific human antibodies was cloned from memory B-cells derived from healthy elderly human donor cohorts and recombinantly expressed and characterized. Additionally, a screening campaign for onal backup antibodies was ted in a cohort of selected presymptomatic patients with Huntington's disease (HD) carrying different lengths CAG repeat expansions.
To further characterize the binding of the fied inant human-derived HTT NI—302 antibodies to e and ated HTT Exonl proteins with 21, 35 or 49 polyQ repeats filter retardation assay utilizing 0.2 ug/ml primary antibody and dot-blot analysis utilizing l ug/ml primary antibody were performed as described in Example 7, supra.
As shown in Fig. 21 and Fig. 32 (A, G), on the dot blot (Fig. left side, DotBlot) most of the antibodies characterized showed a preference for detection of HTT proteins with expanded polyQ tracts (HD49>>HD35>HD21). Furthermore, the signal ity increased with increasing incubation times of the aggregation reactions of HD35 and HD49 in particular for antibodies NI-302.15F9, NI—302.71F6 (Fig. 21, first row ofblots) and -64E5 (Fig. 32 A, left side). In the filter ation analysis NI-302.15F9, NI—302.71F6 (Fig. 21, right side, FRA) and NI64E5 (Fig. 32 A, right side) detected SDS stable HD35 and HD49 aggregates that were retained on the 0.2 um pore size membrane whereas other antibodies such as NI-302.44D7 and NI-302.37C12 (Fig. 21, second row of blots) or NI-302.4A6, NI-302.12H2 and NI- 302.8Ml (Fig. 32 D, E, F) did not bind to aggregates on the filter. Antibody 33Cll served as control in this experiment (Fig. 32 G). These findings based on spotted protein preparations suggest that several of the cloned NI-302 antibodies show a preference for aggregated HTT conformations with pathogenic polyQ expansions.
Additionally, the binding specificity of the identified antibodies to unrelated proteins, in particular to proteins forming aggregates was assessed by direct ELISA (Fig. 22 and Fig. 33 A, D-F) as already described in Example 8, supra. The s showed that most of the human- derived NI-302 antibodies tested bind specifically to HTT with absent g to the other unrelated proteins tested.
Example 26: Assessment of the binding and epitope mapping of human-derived HTT antibodies To map the HTT epitope recognized by the newly identified derived antibodies epitope mapping with synthetic peptides was performed as described above in e 9 (Fig. 23 and Fig. 35 A, D—F). Additionally, the half maximal effective concentration (ECso) of the HTT antibodies to upled peptide fragments of the HTT Exon 1 by direct ELISA with BSA- coupled Htt Exonl domain peptides was determined as well as ECso ination were performed as described in Example 10.
Example 27: Assessment of the binding of HTT antibodies in human HTT transgenic mice The characterization of the binding of the identified antibodies to HTT pathology in human HTT transgenic mouse brain tissues was assessed as described in Example 12, supra with the difference that the tion ofthe sections was performed with 5 nM ffig.3M (74Cl l, 39Cl2, llA4, 22H9, 78Hl2, 37Cl2, 7D8, 72F10) or 50 nM trations (15F9, 71F6, 55D8, 44D7, 7A8, 64E5) ofthe anti-HTT antibodies. As shown in Fig. 24 the identified human—derived anti- HTT antibodies revealed a prominent and highly specific staining of neuronal intranuclear inclusion pathology in the striatum and cortex of R6/1 transgenic animals, as also shown for the antibodies .33C11, NI-302.63F3, and NI—302.35Cl, described above. These findings are consistent with the high affinity binding to HTT aggregates as determined by ELISA and dot blot analysis in Example 26.
Example 28: Basic characterization of transgenic mouse model R6/1 of Huntington's disease (HD) Tg(HDexon1)61pr/J transgenic mice harbor a 1.9 kb transgene which was isolated from a phage genomic clone derived from an HD patient and contained the 5' end of the human huntingtin (HTT) gene. It was composed of approximately 1 kb of 5' UTR sequences, exon 1 (carrying ed CAG repeats of ~130 units) and the first 262 bp of intron 1. This uct was microinjected into single cell CBAxC57BL/6 embryos. Male founder R6 was bred to CBAxC57BL/6 females, producing several founder lines (Mangiarini et al., Cell 87 (1996), 493—506). Mice from founder line R6/l have the ene integrated as a single intact copy which is ubiquitously expressed. Transgenic mice on a mixed CBAxC57BL/6 genetic ound were backcrossed to C57BL/6J for more than 12 to generate the congenic strain B6.Cg-Tg(HDexon1)6lpr/J. R6/l transgenic mice exhibit a progressive neurological phenotype that mimics many of the features of HD, including choreiform—like movements, ntary stereotypic movements, , and epileptic seizures, as well as nonmovement er components, including unusual vocalization. They urinate frequently and exhibit loss ofbody weight and muscle bulk through the course of the disease. Neurologically they develop neuronal intranuclear inclusions (N11) which contain both the HTT and ubiquitin proteins.
These N11 have also been identified in human HD patients. The age of onset of HD symptoms is reported to occur between 15 and 21 weeks for this 6/ 1 line (Naver et a], Neuroscience 122 (2003), 057; Hodges et al, Genes Brain BehaV. 7(3) (2008), 288-299).
R6/l transgenic mice obtained from Jackson Laboratories were expanded and longitudinally characterized with respect to behavior phenotype, longitudinal development of body weight, total brain weight, histopathological is and survival, as shown in Fig. 25. The findings obtained by in large were in line with the published data and identified this mouse line as a le preclinical model for efficacy studies with human-derived NI-302 antibodies ing aggregated HTT.
Example 29: Basic characterization of enic mouse model N171-82Q of HD The B6C3-Tg(HD82Gln)8lDbo/J (N171—82Q) enic mouse line (Schilling er al., Hum Mol Genet. 8(3) (1999), 397-407) is a well characterized mouse model for HD. B6C3- Tg(HD82Gln)8lDbo/J (Nl7l- 82Q) transgenic mice expresses an N—terminally truncated human HTT cDNA that encodes 82 glutamines and encompasses the first 171 amino acids. The altered HTT cDNA is under l of a mouse prion protein promoter. Expression is observed in neurons of the central nervous system. Mice expressing this transgene appear normal at birth h 1-2 months. However, the mice fail to gain weight, develop tremors, hypokinesis and lack coordination. They exhibit an abnormal gait and frequent hind limb clasping. Their life expectancy is 5-6 . Studies using HTT dies indicated diffuse nuclear labeling and numerous immunoreactive nuclear inclusions in le neuron populations. Additionally neuritic damage was evident.
N17l-82Q enic mice obtained from Jackson Laboratories were expanded and longitudinally characterized with respect to behavior phenotype, longitudinal development of body weight, total brain weight at endstage, histopathological analysis and survival (Fig. 26).
These findings by in large were in line with published data and identified this mouse line additionally to the mouse line described in Example 29, as a suitable preclinical model for efficacy studies with human-derived NI-302 antibodies targeting aggregated HTT. e 30: ment of neuronal inclusion ng in Huntington's disease (HD) patients To assess the staining of neuronal ions by the identified antibodies of the present invention in patients immunochistochemical analysis was performed. The assessment of the binding of identified antibodies to HTT pathology in human brain tissues was assessed as described in Example 12, supra with the difference that the incubation of the ns was performed with 50 nM ofNI-302.33C11, 50 nM ofNI-302.63F3 or 100 nM ofNI-302.35Cl antibody.
As shown in Fig. 27 the immunohistochemical analysis with the polyP-region binding antibody NI-302.33C11 showed a staining of neuronal intranuclear inclusions in al s of Huntington Disease patients (Fig. 27 A—D) at 50 nM and in striatal neurons of 270 day old, late disease stage B6.Cg-Tg(HDexon1)6leb/J) transgenic animals at 1 (E) and 5 nM (F) concentration, while no staining was detected in ansgenic littermates (G), when the primary antibody was omitted during the staining (H) or if tissue of non-Huntington Disease controls was stained with 50 nM of .33C11. The P-rich—domain dy NI-302.63F3 (Fig. 28) and anti-C-terminal domain antibody NI-302.35C1 (Fig. 29) showed within the immunohistochemical analysis with 50 nM of NI-302.63F3 or 100 nM of NI—302.35C1 a staining of neuronal uclear inclusions (A—C) and staining of some neurites (D) of cortical neurons of four different Huntington Disease patients (A-D). A staining could also be observed in the striatal neurons of 270 day old, late disease stage B6.Cg-Tg(HDexonl)6leb/J) transgenic animals at 1 (E) and 50 nM (F) concentration. No staining was detected in non- transgenic littermates (G), if primary antibody was omitted during the staining (H) or if tissue ofnon-Huntington Disease controls was stained with 50 nM ofNI—302.63F3 or 100 nM ofNI- 302.35C 1, respectively.
In contrast to the specific anti-HTT antibodies of the present invention, immunohistochemical analysis with the commercially ble anti-polyQ antibody Mab1574 0, Chemicon) showed additional staining of tissue, z'.e. a more l nuclear and cytoplasmic staining and staining ofsome neurites (Fig. 30 A, D) ofcortical neurons of four different Huntington Disease ts and in al neurons of presymptomatic, 150 day old (Fig. 30 E) and 270 day old (Fig. 30 F), late disease stage B6.Cg-Tg(HDexonl)6leb/J) transgenic animals.
Example 31: Characterization of binding affinity and selectivity of anti-poly Q/P domain NI—302.7D8 dy utilizing direct ELISA and ECso To determine the half maximal effective concentration (ECso) of recombinant human-derived HTT antibody NI-302.7D8 to soluble and aggregated HTT Exonl proteins with 21 or 49 polyQ repeats direct ELISA and EC50 determination was performed as described in Example 6, supra.
It could be shown that NI-302.7D8 binds with similar high affinity to soluble GST-HDZI and aggregated HD21 with an ECso of approximately 50 to 100 nM albeit showing a preference to the elongated more pathogenic form of ated HD49 and soluble GST-HD49 with an EC50 of 17 and 6 nM respectively (Fig. 31 B) Accordingly, the human-derived HTT oly Q/P domain antibody NI—302.7D8 targets an epitope d in aggregated as well as in an uncut, more linear structure of HTT Exonl n with high-affinity in the low nanomolar range.
Additionally, to characterize the binding of inant human-derived HTT antibody N1- 302.7D8 to soluble and aggregated HTT Exonl proteins with 21, 35 or 49 polyQ repeats using filter retardation assay and dot blot at a tration of lug/ml.as described in Example 7, supra.
It could be shown that on the dot blot (Fig.32 B, left side), antibody NI-302.7D8 equally good detected proteins of huntingtin with expanded polyQ tracts HD35=HD21). In the filter retardation analysis (Fig. 32 B, right side, FRA) N1-302.7D8 did not bind to SDS stable HD21, HD35 or HD49 aggregates on the filter membrane.
Furthermore, to determine the binding of NI-302.7D8 recombinant dy to unrelated aggregating protein targets, direct ELISA was performed as described in Example 8, supra. As shown in Fig. 33 B human-derived NI-302.7D8 bound specifically to HTT while a binding to unrelated proteins could not be shown.
Example 32: Assessment of the binding epitope of the HTT antibodies NI—302-64E5, NI- 8 and NI—302.72F10 To map the huntingtin epitope recognized by the NI64E5, NI-302.7D8 and NI-302.72F 10 derived antibody epitope mapping with synthetic peptides was performed as described above in Example 9.
Figure 35 A shows a prominent binding ofNI64E5 to es number 10 to 12 indicating that the epitope recognized by this antibody is localized in the P-rich repeat domain of gtin. The NI-302.64E5 binding epitope is therefore predicted to be zed within HTT amino acids 48-PQPPPQAQPL-58 (SEQ ID No.: 200). As shown in Fig. 35 B, prominent g of NI-302.7D8 was observed to es number 6 to 8 indicating that the epitope recognized by this dy is localized in the polyP repeat domain of huntingtin. The NI-302.7D8 binding epitope is therefore predicted to be localized within HTT amino acids 28- QQQQQQQPPP-37 (SEQ ID No.: 201). In contrast, prominent binding of NI-302.72F10 was observed to peptides number 15 and 16 indicating that the epitope recognized by this antibody is localized at the anti-N—terminal domain of HTT (Fig. 35 C). The NI-302.72F10 binding epitope was therefore predicted to be localized within HTT amino acids 70- PPPGPAVAEEPLH-82 (SEQ ID No.: 202).
Example 32: Characterization of binding affinity and selectivity of anti-N—terminal domain antibody NI—302.15E8 utilizing direct ELISA and ECso To determine the half maximal effective concentration (ECso) of recombinant human-derived HTT antibody . 15E8 to soluble and aggregated HTT Exonl proteins with 21 or 49 polyQ repeats direct ELISA and ECso determination was performed as described in e 6, supra.
It could be shown that NI-302.15E8 binds with higher affinity to non-aggregated GST—HD49 and GST-HD21 and less affinity to aggregated HD49 and HD21, see Fig. 14 A and B.
Accordingly, the human-derived HTT anti-N—terminal domain antibody NI-302.15E8 target an epitope d in both aggregated as well as in soluble forms of HTT, albeit with a higher affinity to soluble forms of HTT.
Example 33: Epitope mapping by direct ELISA binding to different Exonl peptides of the HTT antibody NI—302.15E8 To determine the half maximal effective tration (EC50) of recombinant human-derived HTT antibody NI-302.15E8 to upled e fragments of the huntingtin Exon 1 direct ELISA with BSA-coupled Htt Exonl domain peptides and ECso determination were performed as described in Example 10.
As shown in Fig. 15 NI-302.15E8 binds with high affinity to the first 19 BSA-coupled amino acids at the N—terminus as well as to full-length GST-HD49 with an ECso of approx. 0.1 or 15, respectively. e 34: Impact ofHTT antibody NI-302.35C1 on behavioral deficits in human HTT transgenic mice An Elevated Plus Maze test to measure anxiety-like behavior and a Pole test to measure motor performance and coordination in human HTT mice were performed to study the anti-HTT dy NI-302.35Cl in viva.
Groups and Treatment For the behavioral analysis two groups of mice with n=24 (12/ 12 male/female) B6.Cg- Tg(HDex0nl)6leb/J transgenic (tg) mice as described in Example 12 and one group of wild type (wt) mice were used. The groups of transgenic mice received intraperitoneal treatment of either 30mg/kg mouse chimeric NI—302.35Cl or vehicle starting at an age for 6-7 weeks until end stage phenotype of the mice between 7 to 9 months of age and the wild type mice were injected with the same volume of vehicle. The Elevated-Plus-Maze and pole test behavioral tests were performed at an age of 16 and 18 weeks of age respectively.
Elevated Plus Maze test The ed Plus Maze test was performed according to Naver et al., science 122 (2003), 1049—1057. The maze was elevated 50 cm above the floor. Four maze arms (30 cm x 5 cm) originated from a central platform forming a cross. Two of the arms d opposite each other were enclosed by 15 cm high walls (closed arms) while the other arms did not have any kind of screening (open arms). The test were performed at the beginning of the dark phase of the animals and the illumination on the open arm was in the range of 401ux. Each mouse was placed in the center of the Plus Maze facing an open arm. The experiment lasted for 5 min and was recorded with a Videotracking system (VideoMot Software, TSE Systems). Between the sessions, the maze was rinsed with water and dried with a paper towel. Subsequently the number of entries made into open and closed arms as well as the time spent in open and closed tments were evaluated. An entry was defined as all four paws in one arm. The number of entries into the open arms and the time spent in the open arms are used as indices ofopen space—induced anxiety in mice.
The pole test is used widely to assess basal ganglia-related movement disorders in mice; see, e. g. ra et a]. J. Neurosci. 73 (1997), 45-48; Sedelis et a]. Behav Brain Res. 125 (2001), 109-125, Fernagut et al., Neuroscience 116 (2003), 1123-1130. Briefly, animals were placed p on top of a vertical wooden pole 50 cm long (1 cm in diameter). The base of the pole was placed in the home cage. When placed on the pole, animals orient themselves downward and descend the length of the pole back into their home cage. On the test day, animals received three trials, and the total time to descend (t—total) were measured. The results of the third trial ofthe day is shown in Fig. 24 B.
As shown in Fig. 34 A NI-302.35C1 treated R6/1 animals spend less time in the open arms, entered the open arms less frequently and did less unprotected head clips on the open arm compared to vehicle treated R6/1 animals. Hence the NI-302.35C1 treated R6/1 mice displayed a more anxious phenotype, comparable to the non-transgenic littermates. Furthermore, as shown in Fig. 24 B NI-302.35C1 treated R6/1 animal showed an improved performance in the pole test compared to vehicle treated R6/1 animals reaching levels similar to non-transgenic animals. In summary, the antibody NI-302.35C1 ofthe present invention has a beneficial impact on behavioral performance and motor-related tasks in human HTT transgenic mice.
Example 35: Sequence alignment of HTT dies The determination ent ty or similarity was performed with the standard ters of the BLASTn program as bed in section "Definitions" of the present invention. As shown in Fig. 36 all antibodies of the t invention are rich of tyrosines in the CDRs.
Example 36: Generation of bispecific TT antibodies The generation of bispecific dies can be performed as generally bed in Brennan; see supra. Starting material for producing bispecific antibodies are intact IgG anti-HTT antibodies of the present invention izing either a polyP-region, a polyP-region, the P-rich—region, the C terminal-region or the N—terminal region of HTT exon 1 protein as described in the es and ized in Figure 20. The antibodies are treated with pepsin for three hours at 37 ° C treated in acetate buffer pH 4.0, to cleave the Fc n ofthe antibody.
The on is stopped by increasing the pH to 8 with Tris buffer. Subsequently the solution is filled up with an equal volume of a mixture of 5, 5'—dithiobisnitrobenzoic acid (DTNB) and incubated with thionitrobenzoate (TNB) for 20 hours at room ature. The molar ratio of the DTNB-TNB mixture is 20:30 being established by incubating a 40 mM DTNB solution with a 10 mM DTT solution for several minutes. After further reduction of the two modified F(ab') fragments with 0.1 mM DTT for one hour at 25 ° C, the thus obtained F(ab')-TNB and F(ab')-SH fragments are hybridized to a bispecific F(ab')2-fragment for l h at 25 ° C. Bispecific F(ab')2-fragments were purified via gel filtration (Superdex 200 column).
Example 37: Characterization of binding affinity and selectivity of bispecific anti-HTT antibodies utilizing direct ELISA and EC50 To determine the half maximal effective concentration (EC50) ofbispecific HTT antibodies to soluble and aggregated HTT Exonl proteins with 21 or 49 polyQ repeats direct ELISA and EC50 determination is performed as described in Example 6, supra. The bispecific HTT antibodies bind with high y to all four species including the aggregated HTT Exonl HD49, equally targeted their respective es exposed in aggregated as well as in soluble forms of HTT with low nanomolar y. Additionally, to characterize the binding of bispecific HTT antibodies to soluble and aggregated HTT Exonl proteins with 21, 35 or 49 polyQ repeats filter retardation assay and dot blot as described in Example 7, supra, are performed. On the dot blot, bispecific HTT antibodies preferentially detect constructs ofHTT with expanded polyQ tracts. Furthermore, the signal intensity increases with increasing incubation times of the aggregation reactions of HD35 and HD49. In the filter retardation analysis bispecific HTT antibodies detect HD35 and HD49 aggregates that are retained on the 0.2 um pore size membrane. Based on their dual specificity to HTT and the previous findings for the binding of the individual antibodies on membrane bound protein preparations it is expected that bispecific HTT antibodies preferentially target aggregated HTT conformations with pathogenic polyQ expansions. Furthermore, to ine the binding of bispecific anti- HTT antibodies to unrelated aggregating protein targets, direct ELISA is performed as described in Example 8, supra. In this context, ific anti-HTT antibodies bind specifically to HTT while a binding to ted proteins may not be shown.
Claims (30)
1. A derived monoclonal anti-huntingtin (HTT) antibody or an HTT-binding fragment, synthetic or biotechnological derivative thereof, which izes an epitope in the P-rich region of the amino acid sequence of exon 1 of the HTT gene, and 5 comprises in its variable region the amino acid ce of the VH and VL region of any one of antibodies NI-302.63F3, NI-302.31F11, NI-302.2A2, NI-302.15D3 or NI- 302.64E5 depicted in: (i) VH sequence: SEQ ID NOs: 5, 13, 17, 135, 164, 166; and (ii) VL sequence: SEQ ID NOs: 7, 15, 19, 101, 103, 105, 107, 111, 113, 137, 168, 170, 10 respectively.
2. The antibody or HTT-binding nt, synthetic or biotechnological derivative thereof of claim 1, which is of the IgG type. 15
3. The antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof of claim 1 or 2, wherein the light chain is kappa ().
4. The antibody or HTT-binding fragment, synthetic or hnological derivative thereof of any one of claims 1 to 3, which is capable of binding a peptide comprising the epitope 20 and/or aggregated forms of HTT exon 1.
5. The antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof of any one of claims 1 to 4 which specifically binds an e in the P-rich-region which comprises the amino acid sequence PQPPPQAQPL (SEQ ID No. 140), 25 PPPQLPQPPP (SEQ ID No. 141), QAQPLLPQPQPPPPP (SEQ ID No. 142), or PPPQLPQPPPQAQPL (SEQ ID No. 143).
6. The antibody or HTT-binding nt, synthetic or biotechnological derivative thereof of claim 5, which r comprises a polypeptide sequence which is heterologous to 30 the VH and VL region or the six CDRs.
7. The antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof of claim 6, wherein the polypeptide ce comprises a human constant domain.
8. The antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof of claim 7, wherein the human constant domain is of the IgG type.
9. The antibody of any one of claims 1 to 8, wherein the antibody has a binding affinity 5 corresponding to an EC50 (half maximal effective concentration) value of ≤ 20 nM, for binding HD49 and an EC50 value of ≤ 40 nM for g HD21.
10. The antibody of claim 9, wherein the EC50 value is ≤ 10 nM for g HD49 and ≤ 10 nM for binding HD21.
11. The dy of claim 9, wherein the EC50 value is ≤ 1 nM for binding HD49 and ≤ 1 nM for binding HD21.
12. The antibody of any one of claims 1 to 11 which is a chimeric murine-human or a 15 murinized dy and/or an antibody fragment selected from the group consisting of a single chain Fv fragment (scFv), an F(ab) fragment, an F(ab') fragment, an F(ab')2 nt, and a disulfide-linked Fv fragment (sdFv).
13. One or more polynucleotide(s) encoding at least the variable region of the heavy and 20 light chain of the antibody of any one of claims 1 to 12.
14. The polynucleotide(s) of claim 13, which is (are) a cDNA.
15. One or more vector(s) comprising the polynucleotide(s) of claim 13 or 14.
16. An isolated host cell comprising the cleotide(s) of claim 13 or 14 or the vector(s) of claim 15.
17. A method for preparing an anti-HTT antibody, a hnological derivative, or 30 immunoglobulin chain(s) thereof, said method comprising (a) culturing the cell of claim 16 and (b) isolating the antibody or immunoglobulin chain(s) thereof from the culture.
18. The antibody of any one of claims 1 to 12, which is a bispecific antibody.
19. The antibody of claim 18, which recognizes two different epitopes on a protein encoded by exon 1 of the HTT gene. 5
20. The antibody of any one of claims 1 to 12, 18 and 19, which is (i) detectably labeled; or (ii) ed to a drug.
21. The antibody of claim 20, wherein the detectable label is ed from the group 10 consisting of an enzyme, a radioisotope, a fluorophore, and a heavy metal.
22. A composition comprising the antibody of any one of claims 1 to 12 and 18 to 21, the polynucleotide(s) of claim 13 or 14, the vector(s) of claim 15, or the cell of claim 16. 15
23. The composition of claim 22, wherein the composition is (a) a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier; or (b) a diagnostic composition. 20
24. The composition of claim 23, wherein the pharmaceutical composition further comprises an additional agent useful for ng diseases and/or symptoms associated with HTT amyloidosis, or wherein the diagnostic composition comprises reagents conventionally used in immuno or nucleic acid based diagnostic methods.
25.5. The ition of claim 23 or 24, wherein the pharmaceutical composition is a vaccine.
26. A kit useful in the diagnosis or ring of disorders associated with HTT amyloidosis, said kit comprising at least one dy or an HTT-binding fragment, 30 synthetic or biotechnological derivative thereof of any one of claims 1 to 12 and 18 to 21 with reagents and/or instructions for use.
27. Use of the antibody or HTT-binding nt, synthetic or hnological derivative thereof of any one of claims 1 to 12 and 18 to 21 in the manufacture of a medicament for the treatment of a disease and/or symptoms associated with HTT amyloidosis or for in in vivo detection of or ing a therapeutic and/or diagnostic agent to HTT in the human body. 5
28. The use of claim 27, wherein said in vivo detection comprises positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR), optical imaging or magnetic resonance imaging (MRI).
29. A method for the treatment of a disease and/or symptoms associated with HTT 10 amyloidosis or for in in vivo detection of or targeting a therapeutic and/or stic agent to HTT in the non-human animal body, which comprises administering the antibody or HTT-binding fragment, synthetic or biotechnological derivative thereof of any one of claims 1 to 12 and 18 to 21 to the non-human animal body. 15
30. The method of claim 29, n said in vivo detection comprises positron emission tomography (PET), single photon emission tomography ), near infrared (NIR), optical g or magnetic resonance imaging (MRI).
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